Androgen treatment in females

ABSTRACT

The present invention is directed to a method of using dehydroepiandrosterone to treat a human female with diminished ovarian reserve. The method includes administering about 25 milligrams three times a day of dehydroepiandrosterone per day to the female for at least four weeks to reduce human embryo aneuploidy. The present invention further is directed to a method of treating a human female with diminished ovarian reserve to improve the female&#39;s diminished ovarian reserve.

This application is a continuation-in-part of application Ser. No.12/575,426, filed on Oct. 7, 2009, Ser. No. 12/610,215, filed Oct. 30,2009, and Ser. No. 12/123,877, filed on May 20, 2008 which is acontinuation-in-part of Ser. No. 11/680,973, filed on Mar. 1, 2007 (nowabandoned), Ser. No. 11/269,310, filed on Nov. 8, 2005 now U.S. Pat. No.7,615,544, and Ser. No. 10/973,192, filed Oct. 26, 2004 (now abandoned).applications Ser. No. 12,123,877, Ser. No. 12/575,426 and Ser. No.12/610,215 are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of reproductive technology.

2. Description of the Related Art

The application of assisted reproductive technology has revolutionizedthe treatment of all forms of infertility. The most common assistedreproductive technology is in vitro fertilization (IVF), in which awoman's eggs are harvested and fertilized with a man's sperm in alaboratory. Embryos grown from the sperm and eggs are then chosen to betransferred into the woman's uterus. Assisted reproductive technology inwomen depends on ovarian stimulation and concurrent multiple oocytedevelopment, induced by exogenous gonadotropins.

Infertile women are often treated with gonadotropin treatments such asgonadotropin-releasing hormone (GnRH) flare protocols. Estrogenpre-treatment with concomitant growth hormone (GH) treatment issometimes used in an effort to try and amplify intra-ovarianinsulin-like growth factor-I (IGF-I) paracrine effect, which isexpressed by granulosa cells and enhances gonadotropin action. However,the clinical utility of combined GH/ovarian stimulation is limited andresponses are not dramatic.

Dehydroepiandrosterone (DHEA) is secreted by the adrenal cortex, centralnervous system and the ovarian theca cells and is converted inperipheral tissue to more active forms of androgen or estrogen. DHEAsecretion during childhood is minimal but it increases at adrenarche andpeaks around age 25, the age of maximum fertility, only to reach a nadirafter age 60. There is evidence to support use of exogenous DHEA toincrease ovulation stimulation in older women who respond poorly togonadotropin treatments.

Women with diminished ovarian function have decreased egg production andthe eggs that are produced usually are of a poor quality. Further, womenwith diminished ovarian function tend to encounter difficulty becomingpregnant with or without IVF and experience long time periods toconception and/or have an increased possibility of miscarriage and/orthe increased possibility of having high number/percentages of aneuploidembryos.

Women with diminished ovarian function have largely been considered tobe untreatable.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method of usingdehydroepiandrosterone to treat a human female with diminished ovarianreserve. The method includes administering about 25 milligrams threetimes a day of dehydroepiandrosterone per day to the female for at leastfour weeks to reduce human embryo aneuploidy.

The present invention further is directed to a method of treating ahuman female with diminished ovarian reserve. The method includesevaluating the female's anti-Müllerian hormone concentration andadministering about 75 milligrams of dehydroepiandrosterone per day tothe female for at least four weeks to the female to improve the female'sdiminished ovarian reserve. The method further includes reevaluating thefemale's anti-Müllerian hormone concentration. When the female'santi-Müllerian hormone concentration has not increased, the methodincludes continuing to administer dehydroepiandrosterone to the femaleuntil the female's anti-Müllerian hormone level when reevaluatedincreases indicating an improvement in the female's diminished ovarianreserve.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing improved ovulation induction with DHEA.

FIG. 2 is a graph showing an increase in the number of fertilizedoocytes resulting from oocytes harvested from women with DHEA treatment.

FIG. 3 is a graph showing an increase in the number of fertilizedoocytes resulting from oocytes harvested from women with at least 4weeks of DHEA treatment.

FIG. 4 is a graph showing an increase in the number of day three embryosresulting from oocytes harvested from women with at least 4 weeks ofDHEA treatment.

FIG. 5 is a chart showing chemical pathways of adrenal function.

FIG. 6 is a graph showing cumulative pregnancy rate of time from initialvisit to clinical pregnancy or censor by DHEA for women with prematureovarian aging.

FIG. 7 is a graph showing cumulative pregnancy rate of time from initialvisit to clinical pregnancy or censor by DHEA for women with diminishedovarian reserve.

FIG. 8 is a graph showing a comparison of miscarriage rates between DHEAtreated infertility patients and 2004 national US IVF data.

FIG. 9 is a graph showing a cross-sectional evaluation of AMH levels incorrelation to time from DHEA initiation.

FIG. 10 is a graph showing levels over time from DHEA initiation inwomen who did and did not conceive.

FIG. 11 is a table showing patient characteristics.

FIG. 12 is a table showing hormone levels among 206 patients with normalbaseline FSH.

FIG. 13 is a table showing oocyte yields among patients reaching IVF.

FIG. 14A is a graph showing as-AMH levels (Anti Mullerian Hormoneng/ml).

FIG. 14B is a graph showing as-FSH levels (Follicle Stimulating HormonemlU/ml).

FIG. 15 is a graph showing the definition of as-AMH (Anti MullerianHormone).

FIG. 16A is a graph showing oocyte yields at different ages and AMHlevels.

FIG. 16B is a graph showing oocyte yields at different ages and AMHlevels.

FIG. 17 is a table showing comparisons of pre- and post-DHEA cycles in25 women with DOR.

FIG. 18 is a table showing effectiveness of DHEA supplementation in IVFpregnancies based on AMH.

FIG. 19 is a figure showing oocyte and embryo counts in an indexpatient.

FIG. 20A is a graph showing cumulative pregnancy rates in women with DORwith and without DHEA supplementation—premature ovarian aging (POA). Thefigure demonstrates cumulative pregnancy rates in DHEA and controlpatients with POA.

FIG. 20B is a graph showing cumulative pregnancy rates in women with DORwith and without DHEA supplementation—diminished over reserve (DOR). Thefigure demonstrates cumulative pregnancy rates in women above age 40years.

FIG. 21 is a graph showing age-stratified miscarriage rates in DHEAsupplemented DOR patient in comparison to national U.S. IVF pregnancies.

FIG. 22 is a graph showing spontaneous pregnancy loss in spontaneous andIVF pregnancies at various AMH levels.

FIG. 23 is a graph showing AMH in POA and DOR patients over time of DHEAexposure.

FIG. 24A is a graph showing trends in patient characteristics of ourcenter's IVF population—retrieval by year and age. Graph A demonstratesmean ages for IVF patients between 2005 and year-to-date 2009.

FIG. 24B is a graph showing tends in patient characteristics of ourcenter's IVF population—percent retrievals by year and age. Graph Bdemonstrates the proportional shift from younger patients (<39 years) toolder women (≧40 years).

FIG. 24C is a graph showing trends in patient characteristics of ourcenter's IVF population—AMH by age category. Graph C demonstrates thatthis age shift is also accompanied by a significant fall in AMH levelsin younger women (ages 31-35 years) and, therefore, increasing DOR inthese younger (POA) patients.

FIG. 25A is a graph showing the percentages of aneuploid embryos in DHEAand control patients.

FIG. 25B is a graph showing the absolute of aneuploid embryos in DHEAand control patients.

FIG. 26 is a graph showing AMH concentrations in correlation to timefrom DHEA initiation.

FIG. 27 is a graph of the progression in individual patient AMHconcentrations between initial and follow-up consultations.

FIG. 28 is a graph showing AMH concentrations over time from DHEAinitiation in women who did and did not conceive.

FIG. 29 is a graph showing a ROC curve of AMH.

FIG. 30A is a graph of AMH level percentages of live births,terminations of pregnancy for aneuploidy, and spontaneous miscarriagesper IVF cycle.

FIG. 30B is graph of AMH level percentages of live births, terminationsof pregnancy for aneuploidy, and spontaneous miscarriages per patient.

DETAILED DESCRIPTION OF THE INVENTION

When attempting in vitro fertilization (IVF), older women produce fewoocytes and yield few normal embryos, even when exposed to maximalgonadotropin stimulation. The decreased ability of older women torespond to ovulation inducing medications is evidence that ovarianreserve declines with age. Even with IVF cycles, older women produce fewoocytes and yield few normal embryos when exposed to maximalgonadotropin stimulation. This change in ovarian responsiveness is knownas diminished ovarian reserve or diminished ovarian function.

To improve the number of eggs, the quality of eggs, the number ofembryos, the quality of the embryos, spontaneous pregnancy rates, IVFpregnancy rates, cumulative pregnancy rates and time to conception, toreduce the miscarriage rates, and to increase the male/female birthratio, DHEA is administered for at least two months to a human female ina therapeutically effective amount. Preferably, the human female is apremenopausal human female. The human female may have diminished ovarianreserve. DHEA may be administered to a human female at a dose of betweenabout 50 mg/day and about 100 mg/day, preferably between about 60 mg/dayand about 80 mg/day, and in one study about 75 mg/day. Further, DHEA maybe administered in a time-release formulation, over the course of theday, or in a single dose. For example, the about 75 mg/day could beadministered in a single dose of 75 mg or could be administered as 25 mgthree times throughout the day. DHEA is preferably administered orally,although DHEA may be administered or delivered via other methods, suchas, but not limited to, intravenously and/or topically. DHEA has astatistically significant effect on the above-mentioned factors afterabout 2 months of use, but its effect may continue to increase to aboutfour months or about 16 weeks, preferably about four consecutive monthsor about 16 consecutive weeks, and further may continue past four monthsof use.

The effects of DHEA increase over time, and may reach peaks afterapproximately four to five months of supplementation. It is suggestedthat peaks may occur at four to five months because this time period issimilar to the time period of a complete follicular recruitment cycle.Further, the effect of DHEA is suggested to reduce chromosomalabnormalities and thus substantially decreasing miscarriage rates inhuman females.

I. Improvements in Ovulation

Treatments with an androgen, alone or in conjunction with otherhormones, increase a woman's response to ovulation induction, measuredin both oocyte and embryo yield. Androgens may be, for example,dehydroepiandrosterone (DHEA) or testosterone. DHEA treatment may be anadjunct to ovulation induction. DHEA taken orally for at least about onemonth, preferably for about four months, before optionally initiatinggonadotropin treatment, may prepare the ovaries for gonadotropinstimulation. A large response may be obtainable by combininggonadotropins and DHEA in treatment for at least about a four monthperiod before an IVF cycle.

Young ovaries are characterized by large numbers of antral follicles anda low rate of atresia. In contrast, older ovaries have few antralfollicles, high rates of atresia and exhibit increasing “resistance” toovulation induction. Older women have decreased oocyte quantity andquality, produce fewer high quality embryos and have lower implantationand pregnancy rates. Most follicular atresia occurs after the primordialfollicle resumes growth but before it is gonadotropin responsive enoughfor recruitment. An induced delay in onset of atresia may salvagefollicles for possible ovulation. Interestingly, such an “arrest” of theatretic process has been noted among anovulatory women with polycysticovary syndrome (PCO). For these women follicles remain steroidogenicalycompetent and show evidence of increased aromatase activity compared tolike-sized follicles from normal ovaries. Follicular hypersecretion ofDHEA, which is typical of PCO, is associated with increased aromataseactivity. The increased yield of oocytes and embryos experienced bypatients undergoing DHEA treatment may correspond to this underlyingphysiological process.

II. Improvements to Cumulative Embryo Score

DHEA use beneficially effects oocyte and embryo quality. The observationthat DHEA treatment is associated with improved cumulative embryo scoresinfers that such treatment leads to improved embryo and egg quality.This suggestion is further supported by strong trends towards improvedeuploidy in embryos and improved pregnancy rates.

DHEA treatment includes administering a dose of between about 50 mg/dayand about 100 mg/day, preferably between about 60 mg/day and about 80mg/day, and in one study about 75 mg/day to a human female.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA has a statisticallysignificant effect on cumulative embryo score after about 2 months ofadministration, but its effect may continue to increase to about fourmonths, or about 16 weeks, and further may continue past four months ofuse.

Cumulative embryo score is determined by scoring day 3 embryos andmultiplying the number of cells in the embryo by the embryo grade.Embryo grade is a judgment of the embryologist on embryo quality from 1to 5. Most good embryos are scored 4, with 5 reserved for exceptionalembryos. The grade is based on the uniformity of the cells, the colorand consistency of the cytoplasm, and the amount of fragmentation.Normal embryos are less than 5% fragmented. A woman with three eightcell embryos each with a grade of four would have a cumulative embryosscore of 96, the product of 3×8×4.

A cumulative embryo score for women prior to DHEA use may have beenabout 34. A cumulative embryo score after DHEA use of at least aboutfour consecutive months may be at least about 90, preferably at leastabout 95, and in one study at least about 98. The increase in cumulativeembryo score may be at least about 56, preferably at least about 60, andin one study about 64. The difference in the cumulative embryo scoreprior to DHEA use and the cumulative embryo score after DHEA use isstatistically significant, p<0.001. The mean increase in embryo scorewas about 57+/−14.7 after about 16.1 weeks of DHEA administration. Assuch, DHEA treatment significantly improves the cumulative embryo score.

III. Increase in the Number of Fertilized Oocytes

DHEA treatment significantly increased the number of fertilized oocytesproduced by women. DHEA treatment includes administering a dose ofbetween about 50 mg/day and about 100 mg/day, preferably between about60 mg/day and about 80 mg/day, and in one study about 75 mg/day to ahuman female. Particularly, the DHEA treatment may be administered to apremenopausal woman with diminished ovarian function. DHEA may have aneffect on the number of fertilized oocytes after about 4 consecutiveweeks. However, DHEA has a significant effect on the number offertilized oocytes after about 8 weeks or about 2 months ofadministration, and its effect may continue to increase to about fourmonths, and further may continue past four months of use. Specifically,DHEA treatment has a statistically significant effect after about atleast 16 weeks or about at least 4 months of administration.

The number of fertilized oocytes produced by women significantlyincreased after at least about 4 months of consecutive DHEA treatment in12 women, even though slight improvements were shown after at leastabout four weeks of consecutive DHEA treatment, as shown in FIG. 3. Asshown in FIG. 3, paired comparisons of fertilized oocytes from womenhaving less than about four consecutive weeks of DHEA treatment to thesame women having at least about four consecutive weeks of DHEAtreatment showed an increase of about 2 fertilized oocytes, or a medianincrease of about 2.5 fertilized oocytes. The number of fertilizedoocytes may show more significant increase after at least about 4 monthsof DHEA treatment, and may show maximal increase after at least abouteight months of DHEA treatment.

IV. Increase in the Number of Day 3 Embryos

DHEA treatment significantly increased the number of day 3 embryosproduced by women. DHEA treatment includes administering a dose ofbetween about 50 mg/day and about 100 mg/day, preferably between about60 mg/day and about 80 mg/day, and in one study about 75 mg/day to ahuman female. Particularly, the DHEA treatment may be administered to apremenopausal woman with diminished ovarian function. DHEA may have aneffect of day 3 embryos after about 4 consecutive weeks. However, DHEAhas a significant effect after about 8 weeks or about 2 months ofadministration, but its effect may continue to increase to about fourmonths, and further may continue past four months of use. Specifically,DHEA treatment has a statistically significant effect after about atleast 16 weeks or about at least 4 months of administration.

The number of day 3 embryos produced by women also may significantlyincrease after at least about four months of consecutive DHEA treatmentin 12 women, even though slight increases may be shown after at leastabout 4 weeks of DHEA treatment, as shown in FIG. 4. All of the day 3embryos included in the study were normal based on their appearance andon the number of cells, i.e. at least four cells. Paired comparisons offertilized oocytes from women having less than about four consecutiveweeks of DHEA treatment to the same women having at least about fourconsecutive weeks of DHEA treatment may show an increase of about 1 day3 embryo, and in the study summarized in FIG. 4, an increase of about 2day 3 embryos. While the number of day 3 embryos produced slightlyincreased after at least 4 weeks of DHEA treatment, more significantincrease occurs after at least about 4 months of DHEA treatment, andmaximal increase may occur after at least about eight months of DHEAtreatment.

V. Increase in the Number of Euploid Oocytes

DHEA may improve the number of euploid embryos and embryo transfers inwomen with diminished ovarian reserve (DOR). Pretreatment with DHEA, forat least about one month, preferably at least about four months, inwomen may increase oocyte and embryo quantity, egg and embryo quality,cumulative pregnancy rates, pregnancy rates with IVF and time topregnancy.

DHEA treatment includes administering a dose of between about 50 mg/dayand about 100 mg/day, preferably between about 60 mg/day and about 80mg/day, and in one study about 75 mg/day to a human female.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a significant effect afterabout 8 weeks or about 2 months of administration, but its effect maycontinue to increase to about four months, and further may continue pastfour months of use. Specifically, DHEA treatment has a statisticallysignificant effect after about at least 16 weeks or about at least 4months of administration.

The prevalence of aneuploidy in embryos, produced through IVF, from 27consecutive IVF cycles in women with DOR who also had undergonepreimplantation genetic diagnosis (PGD) was evaluated. Amongst thosecycles, 19 had entered IVF without DHEA treatment and eight had receivedDHEA supplementation for at least four weeks prior to IVF start.

DHEA treatment may result in higher oocyte numbers (10.4±7.3 vs.8.5±4.6) increasing from about 8.5 to about 10.4. A significantly largernumber of DHEA treated IVF cycles (8/8, 100%) had at least one euploidembryo for transfer than in untreated cycles (10/19, 52.6%; Likelihoodratio, p=0.004; Fisher's Exact Test, p=0.026). Neither absolute numbersof euploid embryos after DHEA nor percentages of euploid embryosdiffered significantly in this case, however, between untreated andtreated patients.

As women age, there is a substantial decline in euploidy rates inembryos produced. Thus, the increase in euploidy results in older womenis dramatic evidence of the effectiveness of DHEA in improving embryoquality, and pretreatment with DHEA of women with DOR may significantlyincrease their chances for the transfer of at least one euploid embryo.

VI. Improvements to Ovarian Function

DHEA may have beneficial effects on ovarian function and oocyte andembryo quality. DHEA substitution may rejuvenate certain aspects ofovarian function in older ovaries. Since DHEA declines with age to avery significant degree, intraovarian DHEA deficiency may be causallyrelated to the ovarian aging process.

FIG. 5 shows the pathways for normal adrenal function. As shown in FIG.5, the adrenal enzyme 17,20-desmolase may be responsible for theconversion of 17-hydroxy pregnenolone into DHEA (and the conversion of17-hydroxyprogesterone into androstenedione) which, based on thetwo-cell two-gonadotropin theory, may serve in the ovary as a precursorsubstrate for estradiol and androgens. A patient (Patient B), describedfurther in Example 5 herein, with abnormal 17,20-desmolase (P450c17)function may have a hormone profile characterized by persistently lowDHEA, androstenedione, testosterone and estradiol levels, but normalaldosterone and cortisol levels. Patient B exhibited some of theclassical signs of prematurely aging ovaries which include ovarianresistance to stimulation, poor egg and embryo quality and prematurelyelevated FS H levels.

The decrease in DHEA levels with advancing female age may be an inherentpart of the ovarian aging process and may, at least in part, and on atemporary basis, be reversed by external DHEA substitution. This casedemonstrates that low DHEA levels are, indeed, associated with all theclassical signs of (prematurely) aging ovaries. While association doesnot necessarily suggest causation, the observed sequence of events inthis patient supports the notion that low DHEA levels adversely affectovarian function.

Patient B was initially thought to have largely unexplained infertility.Approximately 10 percent of the female population is believed to sufferfrom premature aging ovaries and this diagnosis is often mistaken forunexplained infertility (Nikolaou and Templeton, 2003, Gleicher N,2005). Patient B later developed signs of prematurely aging ovaries and,finally, showed elevated FSH levels. In the time sequence in which allof these observations were made, Patient B followed the classicalparallel premature aging curve (Nikolaou and Templeton, 2003; GleicherN, 2005).

Once substituted with oral DHEA a reversal of many findingscharacteristic of the aging ovary was noted. DHEA treatment includesadministering a dose of between about 50 mg/day and about 100 mg/day,preferably between about 60 mg/day and about 80 mg/day, and in one studyabout 75 mg/day to a human female. The DHEA dose could be administeredas a single dose or as multiple doses over the course of a day.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a significant effect afterabout 8 weeks or about 2 months of administration, but its effect maycontinue to increase to about four months, and further may continue pastfour months of use. Specifically, DHEA treatment has a statisticallysignificant effect after about at least 16 weeks or about at least 4months of administration.

After DHEA administration, Patient B's DHEA and dehydroepiandrosteronesulfate (DHEAS) levels normalized. In subsequent natural cycles anapparently normal spontaneous follicular response was observed, withnormal ovulatory estradiol levels in a patient with persistently lowestradiol levels before DHEA treatment.

DHEA deficiency may be a cause of female infertility and may be apossible causative agent in the aging processes of the ovary. The casestudy involving Patient B also presents further confirmation of thevalue of DHEA substitution whenever the suspicion exists that ovariesmay be lacking of DHEA substrate. Since the process is familial(Nikolaou and Templeton, 2003), it is reasonable to assume that, likeother adrenal enzymatic defects, 17,20-desmolase deficiency may occureither in a sporadic or in an inherited form. As both forms will resultin abnormally low DHEA levels, both may lead to phenotypical expressionas premature ovarian aging.

VII. Increase in Spontaneous Conceptions

Additionally, with DHEA treatment, there may be an unexpectedly largenumber of spontaneous conceptions in women waiting to go into an IVFcycle. DHEA treatment includes administering a dose of between about 50mg/day and about 100 mg/day, preferably between about 60 mg/day andabout 80 mg/day, and in one study about 75 mg/day to a human female.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a more significant effectafter about 8 weeks or about 2 months of administration, but its effectmay continue to increase to about four months, and further may continuepast four months of use. Specifically, DHEA treatment has astatistically significant effect after about at least 16 weeks or aboutat least 4 months of administration.

The DHEA treatment may be at least about 2 weeks before spontaneousconception occurs. In the population of women who are waiting to go intoIVF, the spontaneous pregnancy rate is a fraction of about 1% per month.However, in the population of women who have been on DHEA treatment,there were 13 spontaneous pregnancies out of 60 women. As such, DHEAtreatment increases spontaneous pregnancies in one study at least about21 fold. This provides evidence that DHEA works not only in conjunctionwith gonadotropin stimulation of ovaries, but also without gonadotropinstimulation of ovaries.

VIII. Increase in Male Fetus Sex Ratio

A further effect of DHEA treatment is raising androgen levels in afemale to increase the male fetus sex ratio. The gender of offspring maynot be solely determined by chance. More highly androgenized femalemammals give birth to more male offspring. Androgens, such as DHEA, maybe utilized and an elevated baseline level of above about 250 ng/dl,preferably above about 350 ng/dl, may be sufficient. Infertile womenwith diminished ovarian reserve established a human model to investigatethis theory. Data obtained from this model support an effect ofandrogenization on gender not through a follicular selection mechanismbut rather through different mechanisms than previously theorized asevidenced by occurring after the preimplantation embryo stage.

Routine treatment protocol involves administering about 25 mg ofmicronized, pharmaceutical grade DHEA, TID, to a human female touniformly raise levels of unconjugated DHEA above 350 ng/dl and,therefore, raise baseline testosterone. In six pregnancies spontaneouslyconceived, the distribution between female and male offspring was equal,at three and three, respectively. In contrast, in the remaining 15offspring, which were products of pregnancies achieved through IVF, thedistribution was 12 males and 3 females (p=0.035). Amongst womenundergoing IVF and PGD, 53 embryos were analyzed from 17 IVF cycles, allhaving undergone ICSI. The gender distribution was not significantlyskewed, with 27 being male and 26 female.

The data, demonstrating a strong trend towards both significance overalland significance (p=0.035) amongst IVF patients, suggest that genderdetermination may be influenced through hormone environments. The evendistribution of gender (27 male and 26 female) in this group of patientsargues against a selection process towards male, which is driven by thefollicular environment, as has been previously suggested. The evendistribution of gender in preimplantation embryos, seen in the controlgroup, also speaks against such an effect.

The only remaining conclusion from the here presented data is thatfemale androgenization affects gender selection after thepreimplantation embryo stage and that, by definition, identifies thestage of androgenic influence on gender at or after implantation. Allbut one IVF cycles in study and control groups underwent ICSI, whichrequires the removal of granulose cells from the oocyte. One hypothesisis that such a removal may render the local environment more favorabletowards the implantation of male than female embryos. A secondhypothesis would suggest a similar effect, based on the difference inhormonal milieu in the luteal phase between IVF and spontaneousconception cycles, with the former uniformly supported by progesteroneand the latter only sporadically, or not at all. The data providesevidence that the androgenization of females may increase the prevalenceof male offspring, especially with IVF.

IX. Increase in Pregnancy Rates

An additional benefit of DHEA treatment is an unexpectedly high numberof pregnancies in women, particularly in women with diminished ovarianfunction. DHEA supplementation is also associated with increasedcumulative pregnancy rates and a shorter interval to pregnancy amongwomen with evidence of decreased ovarian function entering evaluationand treatment for infertility.

DHEA treatment includes administering a dose of between about 50 mg/dayand about 100 mg/day, preferably between about 60 mg/day and about 80mg/day, and in one study about 75 mg/day to a human female. Further,DHEA may be administered in a time-release formulation, over the courseof the day, or in a single dose. For example, the about 75 mg/day couldbe administered in a single dose of 75 mg or could be administered as 25mg three times throughout the day. Particularly, the DHEA treatment maybe administered to a premenopausal woman with diminished ovarianfunction. DHEA may have an effect after about 4 consecutive weeks.However, DHEA has a significant effect after about 8 weeks or about 2months of administration, but its effect may continue to increase toabout four months, and further may continue past four months of use.Specifically, DHEA treatment has a statistically significant effectafter about at least 16 weeks or about at least 4 months ofadministration.

A case control study of 190 women over 30 years old with diminishedovarian function were studied between 1999 and December 2005. The studygroup included 89 patients with a mean age of about 41.6 who usedsupplementation of about 75 mg daily of oral, micronized DHEA for up tofour months prior to entry into IVF. The control group composed 101patients with a mean age of about 40.0 who received infertilitytreatment but did not use DHEA. The primary outcome was clinicalpregnancy after the patient's initial visit.

Ovarian stimulation was identical for study and control groups andconsisted of microdose agonist flare, followed by maximal dosagegonadotropin stimulation, using about 300-450 IU of FSH and about 150 IUof HMG. Study patients received DHEA continuously until a positivepregnancy test was obtained or until the patient dropped out oftreatment.

Using a developed Cox proportional hazards model, the proportionalhazards of pregnancy among women using DHEA was compared with thecontrols group. The results were that cumulative clinical pregnancyrates were significantly higher in the study group (25 pregnancies of 89patients for 28% vs. 11 pregnancies of 101 patients for 11%; relativehazard of pregnancy in study group (HR 3.8; 95% CI 1.2 to 11.8;p<0.05)). Specifically, about 28% of the patients that received DHEAachieved a clinical pregnancy, and about 11% of the patients that didnot receive DHEA achieved clinical pregnancy. As such, DHEA treatmentincreases the percentage of clinical pregnancies between about 130% andabout 180%, preferably between about 140% and about 170%, and in onestudy about 157%. As such, DHEA treatment increases clinical pregnanciesby at least about 150%.

Further, the results of this study show a statistically significantpercentage of women that achieved clinical pregnancy only with DHEAtreatment. See Table 8 in Example 7 herein. Table 8 shows 25 of 89 womenin the DHEA treated group achieving clinical pregnancy, including 6 of16 with no other treatment other than DHEA, and 6 of 9 women hadintrauterine insemination (IUI/COH) but no IVF. About at least one-halfof the patients (or at least about 50% of the patients), a total of 12out of the 25 women (about 6 of 16 women with no other treatment, andabout 6 of 9 women treated with intrauterine insemination) thatestablished pregnancy did so spontaneously (i.e., with no IVFtreatment). As such, DHEA treatment also increases the percentage ofclinical pregnancies and significantly reduces the cumulative time topregnancy.

Along with increased clinical pregnancies, women in this study, with amean age of about 41.6, which were treated with DHEA had decreasedmiscarriage rates. Specifically, approximately 36% of the women in thecontrol group (4 of 11 women) that did not receive DHEA had miscarriagesand, in comparison, only approximately 20% of the women in theDHEA-treated group (5 of 25 women) had miscarriages. As such, DHEAtreatment decreased the miscarriage rate between about 30% and about60%, preferably between about 40% and about 50%, and in one study about44%. DHEA treatment decreases the miscarriage rate by at least about ⅓,and preferably by at least about ½.

The data, described further herein, provides evidence that the DHEAsupplementation improves spontaneous pregnancy rates, IVF pregnancyrates, cumulative pregnancy rates, and decreases the time interval topregnancy.

X. Decrease in Miscarriage Rates

Supplementation with dehydroepiandrosterone (DHEA) as described hereinbelow decreases miscarriage rates in infertile women with diminishedovarian reserve. DHEA administration, for an average of at least 2months, decreases the miscarriage rate. DHEA treatment includesadministering a dose of between about 50 mg/day and about 100 mg/day,preferably between about 60 mg/day and about 80 mg/day, and in one studyabout 75 mg/day to a human female. Further, DHEA may be administered ina time-release formulation, over the course of the day, or in a singledose. For example, the about 75 mg/day could be administered in a singledose of 75 mg or could be administered as 25 mg three times throughoutthe day. Particularly, the DHEA treatment may be administered to apremenopausal woman with diminished ovarian function. DHEA may have aneffect after about 4 consecutive weeks. However, DHEA has a moresignificant effect after about 8 weeks or about 2 months ofadministration, but its effect may continue to increase to about fourmonths, and further may continue past four months of use. Specifically,DHEA treatment has a statistically significant effect after at leastabout 16 weeks or at least about 4 months of administration, andpreferably, DHEA treatment is administered for at least about 16consecutive weeks or at least about 4 months.

About 85% of miscarriages are due to chromosomal abnormalities. As such,decreasing the miscarriage rates in women may indicate a decrease inaneuploidy rates.

After about at least two months of prior DHEA supplementation, the rateof clinical miscarriages in 73 pregnancies, established at twoindependent fertility centers in the United States (U.S.) and Canada,was compared to the national U.S. miscarriage rates, reported for invitro fertilization (IVF) pregnancies for the year 2004.

The reduction in miscarriage rates in DHEA pregnancies at both centerswere similar (15.0% and 15.2%) for a combined reduction in miscarriagerates of about 15.1%. The Mantel-Haenszel common odds ratio (and 95% CI)for the odds of miscarriage with DHEA supplementation, stratified byage, was significantly lower relative to the odds of miscarriage in thegeneral U.S. IVF population [0.49 (0.25-0.94; p=0.04)]. Miscarriagerates after DHEA supplementation was lower at all ages than the 2004 USnational averages, but the difference was more pronounced above age 35years.

More specifically, DHEA treatment decreases the miscarriage rate forwomen under the age of about 35 between about 5% and about 25%,preferably between about 10% and about 20%, and in one study about15.7%. DHEA treatment decreases the miscarriage rate for women under theage of about 35 by at least about one-seventh. Further, DHEA treatmentdecreases the miscarriage rate for women between the ages of about 35and about 37 between about 50% and about 70%, preferably between about55% and about 65%, and in one study about 60.8%. DHEA treatmentdecreases the miscarriage rate for women between the ages of about 35and about 37 by at least about one-half. Also, DHEA treatment decreasesthe miscarriage rate for women between the ages of about 38 and about 40between about 20% and about 40%, preferably between about 25% and about35%, and in one study about 31.6%. DHEA treatment decreases themiscarriage rate for women between the ages of about 38 and about 40 byat least about ¼, and preferably by at least about ⅓. Additionally, DHEAtreatment decreases the miscarriage rate for women between the ages ofabout 41 and about 42 between about 30% and about 60%, preferablybetween about 40% and about 50%, and in one study about 45.3%. DHEAtreatment decreases the miscarriage rate for women between the ages ofabout 41 and about 42 by at least about ⅓, and preferably by at leastabout ½. Further, DHEA treatment decreases the miscarriage rate forwomen over the age of about 42 between about 40% and about 60%,preferably between about 45% and about 55%, and in one study about50.1%. DHEA treatment decreases the miscarriage rate for women over theage of about 42 by at least about ½.

DHEA supplementation is associated with a significantly decreasedmiscarriage rate in women, especially above the age of about 35. DHEAtreatment decreases the miscarriage rate for women over the age of about35 by at least about 30% or at least about ⅓. Supplementation with DHEAreduces the miscarriage risk in this high risk population to levelsreported for the general population.

This observation supports a beneficial effect of DHEA on aneuploidyrates. DHEA treated women with diminished ovarian reserve, who producefew embryos, only rarely qualify for preimplantation genetic screening.Data accumulation on embryo aneuploidy rates is, therefore, difficult.Because embryo aneuploidy rates are reflected in miscarriage rates, bydemonstrating a remarkable reduction in miscarriage rates, there iscircumstantial evidence that DHEA supplementation may reduce the rate ofaneuploid embryos in infertile women.

XI. More on Decreasing Miscarriage Rates

Dehydroepinadrosterone (DHEA) supplementation improves pregnancy chancesin women with diminished ovarian reserve (DOR) by possibly reducinganeuploidy. Since a large majority of spontaneous miscarriages areassociated with aneuploidy, one can speculate that DHEA supplementationmay also reduce miscarriage rates.

We retroactively compared, utilizing two independent statistical models,miscarriage rates in 73 DHEA supplemented pregnancies at two independentNorth American infertility centers, age-stratified, to miscarriagesreported in a national U.S. in vitro fertilization (IVF) data base.

After DHEA supplementation the miscarriage rate at both centers was15.1% (15.0% and 15.2%, respectively). For DHEA supplementationMantel-Hanszel common odds ratio (and 95% confidence interval),stratified by age, was significantly lower, relative to odds ofmiscarriage in the general IVF control population [0.49 (0.25-0.94;p=0.04)]. Miscarriage rates after DHEA were significantly lower at allages but most pronounced above age 35 years.

Since DOR patients in the literature are reported to experiencesignificantly higher miscarriage rates than average IVF patients, thehere observed reduction in miscarriages after DHEA supplementationexceeds, however, all expectations. Miscarriage rates after DHEA notonly were lower than in an average national IVF population but werecomparable to rates reported in normally fertile populations. Lowmiscarriage rates, comparable to those of normal fertile women, arestatistically impossible to achieve in DOR patients without assumptionof a DHEA effect on embryo ploidy. Beyond further investigations ininfertile populations, these data, therefore, also suggest theinvestigations of pre-conception DHEA supplementation in normal fertilepopulations above age 35 years.

XII. Improvement in Ovarian Reserve

Our study presents the first objective evidence that supplementationwith dehydroepiandrosterone (DHEA) of women with diminished ovarianreserve (DOR) improves ovarian reserve at all ages.

Our objective was to determine whether supplementation withdehydroepiandrosterone (DHEA) of women, suffering from diminishedovarian reserve (DOR), objectively improves ovarian reserve, based onanti-Müllerian hormone levels (AMH).

120 consecutive women, presenting with DOR were patients in this study.We administered DHEA to each patient to improve ovarian reserve.

DHEA administration, for an average of at least about 1 month, improvesovarian reserve. Preferably, DHEA administration lasts for between about15 days to about 150 days, more preferably between about 25 days and 130days, and in one study between about 30 days and about 120 days (mean 73days±27 days).

DHEA administration also includes administering a dose of between about50 mg/day and about 100 mg/day, preferably between about 60 mg/day andabout 80 mg/day, and in one study about 75 mg/day to a human female.Further, DHEA may be administered in a time-release formulation, overthe course of the day, or in a single dose. For example, the about 75mg/day could be administered in a single dose of about 75 mg or could beadministered as about 25 mg three times throughout the day.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a more significant effectafter about 8 weeks or about 2 months of administration, but its effectmay continue to increase to about four months, and further may continuepast four months of use. Specifically, DHEA treatment has astatistically significant effect after at least about 16 weeks or atleast about 4 months of administration, and preferably, DHEA treatmentis administered for at least about 16 consecutive weeks or at leastabout 4 months.

Our main outcome measure was AMH levels in relationship to DHEAsupplementation over days of DHEA supplementation using linearregression and, in longitudinal evaluation, by examining the interactionbetween days of DHEA treatment and pregnancy success in respect tochanges in AMH levels.

Our results were that AMH levels significantly improved after DHEAsupplementation over time (p=0.002). Age (p=0.007) and length oftreatment (p=0.019) were independently associated with increasing AMH.Women under about age 38 years demonstrated higher AMH levels andimproved AMH proportionally more than older females. Longitudinally, AMHlevels improved by approximately 60 percent from 0.22±0.22 ng/ml to0.35±0.03 ng/ml (p<0.0002). Women who reached IVF experienced a 23.64%clinical pregnancy rate. Those who conceived improved AMH significantlymore than women who did not (p=0.001).

In sum, DHEA supplementation significantly improves ovarian reserve withDOR. Additionally, improvement increases with longer DHEAsupplementation and is more pronounced in younger women under age about38 years.

XIII. Age-Specific Anti-MüLlerian Hormone (AMH): Utility of AMH atVarious Ages

Anti-Müllerian hormone (AMH) is increasingly recognized for betterspecificity in reflecting ovarian reserve (OR) than follicle stimulatinghormone (FSH). Like FSH, AMH, however changes with advancing female age.Normal levels should, therefore, vary at different female ages.

We, therefore, established so-called age-specific (as-) AMH levels infour age groups and investigated whether oocytes number, obtained atIVF, differed based on whether a patient's as-AMH was in as-range, belowit or above it.

AMH demonstrated, once again, its better specificity in comparison toFSH by showing narrower normal ranges at all ages. Moreover, as-AMHallowed for discrimination of oocytes yields at all ages. This studyconfirms AMH as a better reflection of OR in comparison to FSH.Moreover, AMH has the additional advantage of not only being able topredict diminished ovarian reserve (DOR) and low oocytes yields but alsohigh oocytes yield, risk for polycystic ovarian syndrome and ovarianhyperstimulation. It, therefore, appears particularly suitable in theinvestigation of OR in younger women.

Abstract of Age-specific Anti-Müllerian Hormone

We assessed whether age-specific (as-) cut offs for anti-Müllerianhormone (AMH) have higher specificity in reflecting ovarian reserve (OR)than non-age-specific (nas-) AMH values. as-AMH values were defined in778 consecutive infertility patients by establishing as-95% confidenceintervals (CI) of AMH at various ages.

Oocytes yields were then compared at various ages in women with normaland abnormal as-AMH. AMH decreased with advancing female age (p<0.0001),differed significantly in each of four selected age categories (p<0.001)and ranges of as-AMH were at all ages narrower than for as-FSH. In 288women who reached in vitro fertilization (IVF), as-AMH, after adjustmentfor age, was statistically predictive of oocytes yields if abnormallylow (<95% CI) or high (>95% CI). Normal and abnormally elevated as-AMHcombined, demonstrated 5.4-times (95% CI 4.1-6.8) greater oocytes yieldsthan abnormally low as-AMH. Like as-FSH, as-AMH better reflects OR thannas-ovarian reserve testing. In contrast to as-FSH, as-AMH, however,defines risk towards diminished OR (DOR) and high oocytes yields (i.e.,potential hyperstimulation syndrome, OHSS) and, therefore, may be aparticularly useful OR test in younger women in whom DOR is mostfrequently overlooked, and who are at highest risk for OHSS. See atleast Example 10, below.

XIII. Review, Summary and New Findings on Dehydroepiandrosterone (DHEA)Supplementation in Women with Diminished Ovarian Reserve (DOR)

A. Overview

Context: As women above age 40 have become the most rapidly growing agegroup giving birth, treatment of diminished ovarian reserve (DOR) hasassumed GREATER importance. Dehydroepinadrosterone (DHEA)supplementation is increasingly utilized for this purpose. A review ofpublished literature is presented.

Evidence Acquisition: PubMed, Cochrane and Ovid Medline were searchedbetween 1995 and 2009 under the following strategy:[<dehydroepiandrosterone or DHEA or androgens or testosterone> and<ovarian reserve or diminished ovarian reserve or ovarian function>].Bibliographies of relevant publications were further explored foradditional relevant citations.

Evidence Synthesis: In absence of prospectively randomized studies,other study formats offer evidence that DHEA supplementation of womenwith DOR to significant degrees improves ovarian function parameters,increases pregnancy chances and, likely by reducing aneuploidy, reducesmiscarriage rates. DHEA effects increase with length of supplementation.

Conclusions: DHEA effects point towards a revised concept of ovarianaging, which suggests that medications may restore aged ovarianenvironments towards “younger ages,” allowing recruited primordialfollicles to mature at improved environmental conditions. Primordialoocytes, therefore, likely do not age, as currently believed, butovarian environments do. DHEA may, therefore, be only the first, amongstother future drugs, capable of, at least partially, restoring ovarianenvironments for folliculogenesis in women with DOR, in the processreducing aneuploidy, improving pregnancy chances and reducingmiscarriages.

B. Historic Developments

Peter R. Casson and associates, at John E. Buster's group (BaylorMedical College, Houston, Tex.) were the first to suggest therapeuticbenefits from dehydroepiandrosterone (DHEA) supplementation in womenwith diminished ovarian reserve (DOR). This group of investigators had along-standing interest in DHEA and contributed many important, initialobservations, which often were not immediately recognized for theirpotential clinical significance.

They first reported that micronized DHEA offers the potential ofpostmenopausal steroidal replacement, adjunctive to estrogen. In adrenaland ovarian steroidogenesis, DHEA is an intermediate product in theconversion of cholesterol to the sex hormones, testosterone andestradiol. They, however, demonstrated that in postmenopausal women thisconversion is not symmetrical and favors androgens. While testosteroneafter DHEA supplementation increases, estradiol remains low. In furtherexploring androgen deficiency in menopause, they then demonstrated thatDHEA has immunomodulatory effects, an observation now well recognizedand therapeutically explored in treating autoimmune diseases.

The same group later demonstrated that vaginally administered DHEA,while delivering equivalent hormone, substantially diminishesbioconversion in comparison to oral micronized product. They followed upby showing that abnormally low DHEA secretion is potentiated by ovarianhyperstimulation, an observation to be discussed in more detail below.

Returning to DHEA as potential postmenopausal steroid replacement, theydemonstrated that DHEA was well tolerated and increased IGF-1 levels.Recurrent themes of their research were the need to address adrenalcortical changes in aging women, and compensating with DHEAsupplementation.

This work led to the above noted case series of women with poor responseto ovarian stimulation with gonadotropins, in which Casson andassociates reported improvements in ovarian response after DHEAsupplementation. Their rational for this study was the previouslyobserved increase in IGF-1 after DHEA supplementation. Since growthhormone had been suggested to improve oocytes yields via IGF-1, theyspeculated that DHEA may be able to achieve similar effects.

Like other achievements by this group, this small case series wentlargely unnoticed. Even the authors, themselves, did not further followup. It was left to a 43 year old patient at our center, years later, torediscover the paper when searching the literature for remedies that mayhelp her overcome severe DOR and resistance to ovarian stimulation. Shein a first in vitro fertilization (IVF) cycle, for the purpose offertility preservation, had produced only one egg and one embryo, andhad been advised to consider oocytes donation.

The patient, an attorney and banker without medical training, based onreview of the literature, identified various potential remedies toimprove her response to stimulation. She chose DHEA, as she later toldus, because it was the only medication she could purchase withoutprescription and, therefore, without our knowledge. In the United States(U.S.), despite being a mild androgen, DHEA is, paradoxically,considered a food supplement and available over the counter, withoutprescription. See at least Example 1.

To our surprise (and unaware of her DHEA supplementation), the patientsin her second IVF cycle produced three oocytes and three embryos ofexcellent quality. We, therefore, no longer refused further cycles. Sheunderwent a total of nine consecutive IVF cycles, and we reported herextraordinary experience.

FIG. 19 is a graph showing oocyte and embryo counts in an index patient.The patient underwent nine consecutive IVF cycles and increased oocytesand embryo yields from cycle to cycle, starting with one egg and embryo,respectively, and ending up with 17 oocytes and 16 embryos in her ninthcycle. Gonadotropin stimulation was reduced in her last cycle forconcerns about possible ovarian hyperstimulation. The patients advisedus of her DHEA supplementation only after her sixth cycle.

In recognition of this patient's contribution to the DHEA research atour center, going forward, she will be designated as the center's indexpatient. As FIG. 19 demonstrates, she from cycle to cycle increasedoocyte and embryo yields. In her ninth cycle, by now 44 years old, hergonadotropin dosage had to be reduced because of concerns abouthyperstimulation. In that cycle, 17 oocytes were retrieved and 16embryos were produced. To assess potential pregnancy chances better,preimplantation genetic diagnosis (PGD) was performed to determine thedegree of aneuploidy in her embryos. Amongst 10 embryos nine werereported aneuploid. The one euploid embryo was cryopreserved.

It was not until after the patient's sixth IVF cycle that she made usaware of her DHEA supplementation. By that point we were wondering how awoman in her mid-40s, from cycle to cycle, could improve oocyte andembryo yields to such a degree. Once informed about the DHEAsupplementation, we initiated a structured clinical investigation ofDHEA supplementation in women with DOR.

An attempt at prospectively randomizing patients had to be abandoned forlack of recruitment. Women with DOR almost uniformly refusedrandomization (trial number NCT00419913). Considering that such patientsoften have limited time left to conceive, this should not surprise.European colleagues, initially convinced they would be able to recruitbetter, attempted randomization in a multi-center effort, in cooperationwith our Center. This trial involved IVF centers in Austria, Switzerlandand the Czech Republic, and also had to be abandoned for lack ofrecruitment. As of this point no prospectively randomized study of DHEAsupplementation in women with DOR has been reported. Best availableevidence, therefore, so far relies on other study formats thanprospectively randomized trials. Available DHEA data, as of this point,are limited to observational, cohort and case control studies. Those arereviewed in the following section.

C. Reported Clinical Experiences

Increase in Oocytes and Embryo Yields

Casson and associates, in their initial report, did not outright suggesta DHEA benefit on DOR. Instead, they claimed that DHEA supplementationmay augment ovarian stimulation with gonadotropins in poor respondersand results in improved oocytes yields. This conclusion was reached insix IVF cycles based on investigation of only five proven poorresponders, under the age 41 years, and with baseline folliclestimulating hormone (FSH) under 20 mIU/ml. After receiving 80 mg ofmicronized DHEA for two months, all study subjects demonstrated improvedresponsiveness in comparison to a prior unsupplemented cycle,characterized by increased peak estradiol and improved peakestradiol/gonadotropin dosage ratios. In addition, one patient delivereda twin pregnancy.

Likely due to the small study size and the chosen study format, thispaper received no follow up attention. The next published report on DHEAsupplementation appeared a full five years later and described ourexperience with the earlier noted index patient. Like Casson et al, we,too, were, first and foremost, impressed by the observed improvement inoocytes yields, which seemed far greater than initially reported by theBaylor group. Indeed, considering the length of observation and numberof repeat cycles in our index patient (FIG. 19), we felt that thelongitudinal observation of this single patient offered even strongersupport for a positive DHEA effect on oocytes numbers. A statisticalerror, like return to median, in our observation seemed less likely thanin an observational study, where patients, in only two observations,served as their own controls.

We were also impressed by the continuous improvement in oocyte (andembryo) numbers with increasing length of DHEA supplementation andspeculated about possible causes: DHEA over time could have cumulativebenefits and/or could have synergistic effects with gonadotropinstimulation, which our index patient underwent practically month aftermonth in pursuit of nine consecutive cycles. Cumulative effects overtime would suggest a DHEA effect on follicular recruitment cycles intheir total length, while synergistic effects with gonadotropinstimulation appeared a possibility based on the Baylor group's reportthat gonadotropins augment adrenocortical DHEA (sulfate) secretion.

More importantly, however, we started to view DHEA supplementation nolonger as just a potential tool in overcoming ovarian resistance tostimulation and increase oocytes yields, but as a potential remedy topositively affect ovarian reserve (OR).

OR is a widely held concept, which assumes that a woman's OR isreflective of chances for conception. In principle, OR is defined by thesize of the remaining follicular pool within ovaries but, in parallel,also assumes a qualitative component.

The new focus on OR represented a significant conceptional changebecause it suggested that DHEA may not only impact oocyte and embryonumbers but also oocyte and embryo quality. It was this consideration,which led towards investigations of egg and embryo quality and,ultimately, of pregnancy success.

Improvements in Oocytes and Embryo Quality

The first 25 DOR patients supplemented with DHEA at our center, inpaired analysis of pre- and post-DHEA cycles, once more confirmedstatistically significant increases in oocytes and embryo numbers. Thisstudy, however, for the first time, also demonstrated that DHEA improvesto significant degrees embryo quality parameters, including embryogrades and average embryo scores. Most importantly, however, this studyfor the first time presented evidence that DHEA significantly increasestransferred embryo numbers.

Since in women with severe DOR the number of embryos available fortransfer is almost always inadequately low, that DHEA could improveembryo transfer numbers suggested that DHEA also may positively affectpregnancy rates.

FIG. 17 is a table showing comparisons of pre- and post-DHEA cycles in25 women with DOR*. *=25 patients were evaluated in their respective IVFcycle outcomes pre- and post-DHEA. This study design potentially biasesoutcome against positive DHEA effects since patients who entered DHEAsupplementation after a prior failed IVF cycle, quite obviously,reflected, in view of their prior IVF treatment failure a negativelyselected patient population. Pre- and post DHEA cycles occurred at ages39±0.8 and 40.4±0.8 years, respectively, also mildly biasing the studyagainst positive DHEA findings. Post-DHEA patients were onsupplementation 17.6±2.13 weeks by time of second IVF cycle. Theuniformity of results of this study, all suggesting quantitative andqualitative IVF outcome improvements (FIG. 17), therefore, stronglyencouraged the center's continuous research efforts.

Improvements in Pregnancy Rates

Aside from abstracts, the next publication was a case controlled studyinvolving 89 DOR patients, prior to IVF, for up to four months,supplemented with DHEA. One-hundred-and-one infertile DOR patients,without DHEA supplementation, served as historical controls. The primarypurpose of this study was to assess potential effects of DHEA onpregnancy rates.

Despite significantly older age (41.6±0.4 vs. 40.0±0.4 years) of DHEApatients, this study for the first time demonstrated that DHEA improvestime to pregnancy and overall pregnancy chances. Cumulative clinicalpregnancies after DHEA (28.1%) were significantly higher than incontrols (10.9%; 95% CI 1.2-11.8; p<0.05). These results were obtainedeven though controls were prognostically a more favorable patient group.

They produced more oocytes (p<0.01), normal day-3 embryos (p<0.05) andeven received more embryos at time of transfer (p<0.05). Clinicalpregnancies were, yet, still significantly higher amongst DHEAsupplemented women.

To us this observation suggested primacy of egg and embryo quality overegg and embryo quantity and, going forward, this paradigm became aguiding principle in how to prepare and stimulate DOR patients for IVF.

Expanded and successful DHEA utilization world-wide is also documentedby quite a number of published abstracts Likely the largest experiencehas been accumulated in Toronto, Canada, by Ed Ryan and his team, whoreport significantly improved clinical pregnancy rates in hundreds ofIVF and insemination cycles, using varying ovarian stimulation protocols(Ryan E, Personal communication, 2009).

In cooperation with Robert F Casper's group at Toronto's Mount SinaiHospital, they recently reported on 47 patients with prior clomiphenecitrate failures who were supplemented with 75 mg daily of DHEA for atleast 60 days prior to inseminations, with stimulation by eitherclomiphene citrate or letrozole in combination with FSH. Controls were46 women, matched by age and baseline FSH without DHEA supplementation.DHEA patients demonstrated significantly higher antral follicle counts,significantly improved pregnancy rates (29.8 vs. 8.7%; CI 1.3-14.8) andlive births (21.3% and 6.5%, respectively), numbers remarkably similarto those reported by our group.

We are also aware of, still unpublished data sets, from Israel, Turkeyand Japan, which all uniformly suggest treatment outcome improvementsafter DHEA supplementation. Conversely, we are unaware of any data setsthat failed to demonstrate such benefits.

Premature Versus Physiologic DOR

With an increasing size data set, it became possible to separate DORpatients into women with age-dependent DOR and younger females withso-called premature ovarian aging (POA). Based on age-specific FSH, wedefined POA as abnormally elevated FSH under age 40 years, andconsidered every woman above age 40 to automatically suffer fromphysiologic, age-dependent DOR.

FIG. 20A is a graph showing cumulative pregnancy rates in women with DORwith and without DHEA supplementation—premature ovarian aging (POA). Thefigure demonstrates on the left side cumulative pregnancy rates in DHEAand control patients with POA (for definition see text). Both patientpopulation demonstrate similar treatment benefits for DHEA, though POApatients appear to have a slight pregnancy advantage, further confirmedin later data presentations. FIG. 20B is a graph showing cumulativepregnancy rates in women with DOR with and without DHEAsupplementation—diminished over reserve (DOR). The right side of thefigure demonstrates cumulative pregnancy rates in women above age 40years.

DHEA supplementation proved similarly effective in both groups, thoughPOA patients, as FIG. 20 demonstrates, do mildly better. The figure alsodemonstrates that beneficial effects of DHEA increase with increasinglength of DHEA supplementation since discrepancies in cumulativepregnancy rates between DHEA and control patients increase with time.

These data confirm observations originally made in the index patient:DHEA effects are relatively quick but do not peak for months. This ledus to require at least six weeks of DHEA supplementation prior to IVFcycle starts. We, however, if clinical circumstances allow, do nothesitate to extend this time period, especially in younger women, tothree to four months. Considering the severity of DOR in DHEAsupplemented patients, we observed surprising numbers of spontaneouslyconceived pregnancies during this waiting period.

Premature Ovarian Failure (POF)

Women who suffer from POA/DOR are distinct from women in outrightpremature ovarian failure (POF), or primary ovarian insufficiency (POI),an acronym recently increasingly applied to this condition. As abovesummarized, until recently, DHEA was only investigated in POA/DORpatients. At our center all successfully treated patients had baselineFSH levels below 40.0 mIU/ml.

Mamas and Mamas, from Athens, Greece, however, recently reported a caseseries of five alleged POF/POI patients, who succeeded in spontaneouslyconceiving after DHEA supplementation.

While intriguing in concept, this report has to be viewed with caution.Not only is this case series very small, but three of the five reportedpatients do not qualify for the diagnosis of POF/POI under standarddefinitions and, likely, resemble previously described POA/DOR patients.

In a brief review Mamas and Mamas more recently reiterated their claim,though without much additional detail. In a personal communication, oneof the authors advised us that they observed additional spontaneouspregnancies in DHEA supplemented POF/POI patients (Mamas L, Personalcommunication, ESHRE Annual Meeting, Amsterdam, The Netherlands, July2009). Our center has registered and initiated a prospectivelyrandomized study of DHEA supplementation in POF/POI patients (trialnumber NCT00948857) but has so far, in a very small number of patients,not yet observed a pregnancy.

This study welcomes collaborating centers and/or referrals of patients.Study participation is free of charges to patients.

Effects on Embryo Ploidy, Miscarriage Risk and Live Birth Rates

We noted earlier that our index patient gave us in her last IVF cyclethe opportunity to investigate 10 of her embryos for aneuploidy. Amongstthose, only one was found euploid. Recognizing current limitations toaccurate preimplantation genetic screening (PGS), we have had limitedopportunities to perform PGS in women with DOR. They usually produceonly small embryo numbers and, in our opinion, therefore, are notqualified for PGS.

In a small pilot study we, however, in 2007 noted that 100 percent ofDHEA treated but only 53 percent of control IVF cycles gave us at leastone euploid embryo (p<0.05). These results were obtained, even thoughDHEA treated patients were older than controls and, therefore, expectedto have more aneuploidy.

Though this difference reached statistical significance, the number ofcases available for investigation was too small to reach a statisticallyrobust enough conclusion that DHEA, indeed, beneficially affects embryoploidy. Because larger patient numbers appeared unlikely in theforeseeable future, we decided to seek alternatives to explore thisquestion further. The close statistical association between embryoaneuploidy and spontaneous pregnancy loss appeared suited for furtherinvestigation. An opportunity presented itself when Ed Ryan, MD(Toronto, Canada), unannounced, offered his center's DHEA data for jointanalysis. Combined, our two centers had produced large enough post-DHEApregnancy numbers to allow for a statistically robust analysis ofmiscarriage rates. Since approximately 80 to 85 percent of allmiscarriages are the consequence of chromosomal abnormalities, weconcluded that a positive DHEA effect on ploidy should be statisticallyreflected in lower miscarriage rates.

A since published study, indeed, confirmed this hypothesis. DHEApregnancies demonstrated significant reduction in spontaneous pregnancyloss in comparison to national U.S. IVF pregnancy rates. Depending onstatistical method utilized, the observed decline in miscarriages was inthe range of 50 to 80 percent.

Additional observations even further strengthened these findings: [1]Miscarriage rates in Toronto and New York were practically identical(15.2 and 15.0%, respectively). [2] In contrast to DHEA patients, whouniformly suffered from DOR, the U.S. national IVF registry reflects aDOR diagnosis in only a small minority of patients. Since DOR patientsdemonstrate significantly higher miscarriage rates than otherinfertility patients, the national control population was stronglybiased against discovery of a DHEA effect on miscarriages. [3] Theobserved combined miscarriage rate of 15.1 percent in DHEA pretreatedpatients mimics spontaneous miscarriage rates reported in normal,fertile populations. [4] The DHEA benefit on miscarriage rates was smallunder age 35 years, but, after that age, progressively increased (FIG.21 is a graph showing age-stratified miscarriage rates in DHEAsupplemented DOR patient in comparison to national U.S. IVF pregnancies.DHEA pretreated patients demonstrated significantly lower miscarriagerates at all ages. The difference was, however, relatively small underage 35 years and progressively increased after that age.).

All of these observations offer strong additional support for theassumption that DHEA, to a significant degree, beneficially affectsage-related miscarriage rates. Such large effects on miscarriage ratesare unachievable unless DHEA beneficially affects ploidy. We, therefore,based on the earlier PGD- and this miscarriage-study, are now convincedthat DHEA beneficially affects embryo ploidy and that it does soincreasingly successfully with advancing female age.

Another study from our center further supports these conclusions (FIG.22). FIG. 22 is a graph showing spontaneous pregnancy loss inspontaneous and IVF pregnancies at various AMH levels. The figuredepicts at various AMH levels in the left column IVF pregnancies (IVF),as previously reported (26), and in the right column spontaneouslyconceived pregnancies (SP). Each column represents 100% of allpregnancies established, separated for live births (black section),voluntary termination of pregnancy (TOP; usually for aneuploidy) andspontaneous miscarriages (SAB). The figure demonstrates that at very lowAMH levels (≦0.40 ng/mL) and at AMH≧1.06 ng/mL, IVF pregnancies led tosignificantly higher live birth rates than spontaneously conceived DHEApregnancies. Lowest pregnancy and live birth rates were observed withIVF and spontaneously between AMH 0.41-1.05 ng/mL, with no spontaneousDHEA pregnancies at all at AMH 0.81-1.05 ng/mL. While in IVF pregnanciesmiscarriage rates were clearly reduced at very low and at higher AMH,miscarriages appeared unaffected (˜50%) in spontaneously conceivedpregnancies.

In that study (FIG. 22), we investigated live birth rates after IVF atextremely low anti-Müllerian hormone (AMH) levels and were surprised howlow miscarriage rates were between non-detectable levels of AMH and 0.4ng/mL. Losses then increased between AMH 0.41-1.05 ng/mL to over 50percent, the expected rate in DOR patients, only to fall, once again, tovery low levels above AMH 1.05 ng/mL.

Since all investigated patients/pregnancies had been pretreated withDHEA, it seems likely that the observed very low miscarriage rates belowAMH 0.4 and above 1.05 ng/mL were the consequence of DHEAsupplementation. These results, however, raised the question why such aDHEA effect would not also be seen at AMH levels 0.41-1.05 ng/mL?

Since submission of those IVF pregnancy data, we had the opportunity toinvestigate 39 spontaneous pregnancies, conceived while on DHEAsupplementation. FIG. 22 demonstrates miscarriage rates in thesepatients in comparison to above noted IVF pregnancies. As the figuredemonstrates, spontaneous DHEA pregnancies in DOR patients, at all lowAMH levels, experience almost identically high miscarriage rates (around50 percent). Spontaneous DHEA conceptions, thus, do not appear tobenefit from DHEA effects on miscarriage rates to the same degree as IVFpregnancies.

This observation suggests two possible explanations: Synergisticgonadotropin stimulation may contribute to the reduction in miscarriagerates observed with DHEA supplementation or women who spontaneouslyconceived simply did not have equal length of DHEA exposure as IVFpatients. We are currently investigating the two explanations.

The possibility of synergistic gonadotropin and DHEA effects issupported by reanalysis of the Toronto groups' previously notedinsemination cycles, stimulated with a clomiphene citrate/letrozole andFSH protocols. Calculating miscarriage rates, we noted rates of 28.5%and 25.3%, respectively, for DHEA and control pregnancies, both ratessignificantly higher, than the 15.2 percent previously reported byRyan's program, mostly involving IVF cycles. Since patients in thisstudy mostly received clomiphene citrate and/or letrozole, these datapotentially favor a synergistically beneficial effect on ovaries betweenDHEA and gonadotropin stimulation, in line with earlier suggestions bythe Baylor group. Final conclusions await, however, furtherinvestigations.

Predicting the Effectiveness of DHEA

FIG. 22 also, once again, suggests special ovarian circumstances at AMHlevels 0.41-1.05 n/mL: between AMH 0.41-0.80 the trend in spontaneousand IVF DHEA pregnancies is towards more miscarriages. Between AMH0.81-105 ng/mL, remarkably, no spontaneous pregnancies were registeredat all. Combined, these observations suggest that at OR level betweenAMH 0.41-1.05 ng/mL beneficial DHEA effects on OR may be less pronouncethan at lower and higher AMH levels. Why that is, remains to bedetermined and is currently under investigation.

Our data on women with severe DOR, thus, suggest that AMH levelsdemonstrate a certain degree of predictability in regards to DHEAutilization.

FIG. 18 summarizes how AMH levels relate to chance of conception andlive births in IVF pregnancies with DHEA supplementation.

FIG. 18 is a table showing effectiveness of DHEA supplementation in IVFpregnancies based on AMH.

As the figure demonstrates, under DHEA supplementation, even in absenceof detectable AMH, an approximate 5 percent pregnancy chance per IVFcycle can be obtained. Even more remarkably, miscarriage rates atundetectable AMH are exceedingly low, thus practically equating clinicalpregnancy and live birth rates. These outcomes remain the same up to AMH0.4 ng/mL, at which point clinical pregnancy chances per treatment cycleapproximately double. Despite higher pregnancy rates, live birth ratesremain, however, unchanged since between AMH 0.41-1.05 ng/mL spontaneouspregnancy wastage is surprisingly high. Above those AMH levels pregnancychances greatly improve and miscarriage risk recedes once again to muchlower levels.

AMH 1.05 ng/mL, thus, represents for DOR patients under DHEAsupplementation a distinct separation point in regards to live birthchances: Up to AMH 1.05 ng/mL the chance of live birth per treatmentcycle is only approximately 5 percent. Above that AMH level, live birthschances are significantly improved.

AMH is, however, also in other ways predictive of treatment success withDHEA. AMH levels increase in parallel to length of DHEA supplementationand this increase is significantly more pronounced in younger POA thanolder DOR patients with physiologically aging ovaries (FIG. 23 is agraph showing AMH in POA and DOR patients over time of DHEA exposure. Asthe figure demonstrates, AMH increases significantly with length of DHEAtreatment (full line). This effect is more pronounce in young POApatients (top line) than older DOR patients (bottom line).). Mostimportantly, however, improvements in AMH levels with DHEAsupplementation are statistically highly predictive of pregnancysuccess.

While these data do not yet allow foreseeing which DOR patient will andwill not conceive under DHEA supplementation, they, combined, can helpoffer patients appropriate informed consents. This is particularlyimportant in view of recently issued ethics guidelines on fertilitytreatments in poor prognosis patients.

Treatment Protocols, Side Effects and Complications

Except for studies by the Baylor group, there are few pharmacologicalstudies, addressing DHEA utilization in reproductive medicine. Thosethat exist, exclusively address postmenopausal women. Since one of theBaylor group's studies induced our index patient to startsupplementation, she, like the Baylor group, supplemented withmicronized DHEA, utilizing an over-the-counter product. In the past,over-the-counter DHEA products have been found inconsistent. Whileproducts may have improved, we have advised against over-the-counterproducts, and have recommended pharmaceutical grade, compounded DHEA, byprescription.

We maintained in all studies and treatment protocols the oral medicationdosage of about 25 mg TID (three times a day for a total of about 75 mgper day), used by our index patient. Others, so far, uniformly have alsoused the same dosage, though this does not mean that it is the bestdosage with least side effects. Studies to determine best dosaging ofDHEA in the treatment of DOR have so far not been performed. There arealso no studies in the literature which compare oral DHEA to otherdelivery systems for the drug in DOR, though studies by the Baylor groupin other patients suggested distinct advantages from micronized andorally delivered DHEA.

Side effects of DHEA supplementation at this dosage are small andprimarily relate to androgen effects. Few patients develop oily skin,acne vulgaris and hair loss but these side effects immediately reverseupon cessation of supplementation. More frequently patients comment onimproved energy levels and sex drives.

In over 1,000 patients supplemented with DHEA so far, we have notencountered even a single complication of serious clinical significance.A recent case report from Israel reported the occurrence of aposttraumatic seizure after one month of DHEA supplementation inattempts to improve oocytes yields. Except for an anecdotal association,there appears, however, no clinical significance to this report. Evenlong-term therapy of DHEA, in dosages similar to the one described here,has been demonstrated to be safe.

As noted earlier, in the U.S. DHEA is, paradoxically, considered a foodsupplement and not a drug, and is, therefore, available withoutprescription. In other developed countries this is not the case. Many,indeed, restrict the compound's availability because of past abuses.DHEA studies reported by our center were until 2007 performed underIRB-approved study protocols. Since 2007 our center has recommended DHEAsupplementation routinely to all patients, diagnosed with POA and/orDOR, since we consider these indications as clinically established. DHEAwas recently listed amongst drugs with “orphan indications” in fertilitytherapy. Patients, nevertheless, still have to sign a DHEA-specificinformed consent, which details potential risk and benefits.

Two other indications for DHEA supplementation are currently still underinvestigation in prospectively randomized, placebo controlled trials:unexplained infertility (trial number NCT00650754) and POF/POI (trialnumber NCT00948857).

How does DHEA Affect OR?

How DHEA improves OR, IVF parameters, pregnancy chances and decreasesmiscarriage rates is, ultimately, still unknown. Previously discussedevidence for beneficial effects on embryo ploidy may, at least in part,explain improvements in miscarriage rates. Assuming improved ploidy, onecan expect more spontaneous pregnancies and pregnancies after IVF sinceit would suggest a pharmacological way of improving embryo selection,though less invasive to embryos than selection via PGS.

Hodges et al suggested that treatments can be developed which willreduce the risk of age-related aneuploidy by influencing meioticchromosome segregation. These investigators believe that majordisturbances in chromosome alignments on the meiotic spindle of oocytes(congression failure), responsible for aneuploidy, result from thecomplex interplay of signals regulating folliculogenesis. They therebyincrease the risk of non-disjunction errors.

DHEA, indeed, may be a first such treatment!

This is a potentially very important concept because it suggests thatthe long held believe that oocytes age and that, therefore, aneuploidyincreases may be incorrect. Instead, this new concept suggests that theunrecruited egg is suspended in time and, likely, does not age to asignificant degree. What causes aneuploidy to increase with age is,therefore, not aging of oocytes but aging of the ovarian environmentwithin which oocytes go through folliculogenesis. By correctingage-related changes in this ovarian environment (declining DHEA levelsis only one amongst many such changes), aneuploidy levels can bemaintained at levels usually only seen in younger women. Reduction ofmiscarriage rates in DHEA pregnancies to those of average, fertilepatient populations is supportive of such a concept.

An effect on all of folliculogenesis (i.e., the whole follicularmaturation cycle) is suggested by a number of already previously notedobservations: the continuous improvement in DHEA effects, seen for atleast five to six months, strongly supports a DHEA effect that increasesas developing follicles are longer and longer exposed to DHEA.Furthermore, AMH is the product of small prenatal follicles. Above notedincrease of AMH levels with length of DHEA exposure (FIG. 23) furthersupports this contention. Finally, we also noted earlier that inspontaneously conceived DHEA pregnancies, miscarriages at different AMHlevels appear mostly unaffected in contrast to pregnancies following IVF(FIG. 22). One of the possible explanations for this observation is theshorter time of DHEA exposure of spontaneously conceived pregnancies.Such an explanation would, of course, also be supportive of the conceptand is further discussed below.

Other potential modes of DHEA action have, however, also to beconsidered: We noted earlier that the Baylor group suspected increasesin ovarian IGF-1 to cause DHEA effects. IGF-1, indeed, appears reducedin poor responders to ovarian stimulation.

There is also increasing evidence that androgens, in general, may(within a therapeutic range) enhance ovarian function. In the mouse,androgens, years back, have been demonstrated to increase follicularrecruitment. Increasing intrafollicular androgen levels augmentgranulose cell AMH and inhibin-B production. Androgen receptors havebeen described in ovarian stroma and granulose cells of primordialfollicles, primary follicles and at more advanced stages offolliculogenesis; and ovarian androgens but not estrogens correlate withsystemic inflammation during ovarian stimulation with gonadotropins.

Frattarelli and associates initially reported that day threetestosterone levels at or under 20 ng/dL were associated with poorer IVFpregnancy rates. They later reported only an association with IVFstimulation parameters but no longer with pregnancy success. Iranianinvestigators, however, recently reported that testosterone levels onday 14 after embryo transfer are predictive of IVF pregnancies. Lossl etal published contradictory papers, one claiming and one refuting thattreatment with aromatase inhibitors (which increases intrafollicularandrogens) improves embryo quality. Contradictory results have also beenreported by French investigators on short-term transdermal testosteroneadministration, with Massin et al reporting no benefit, while Balasch'sgroup in two publications stress the beneficial effects of transdermaltestosterone supplementation on ovarian resistance to stimulation withgonadotropins.

In combination, all of these studies raise the possibility that DHEA maynot be the only androgen that positively affects ovarian functions.Further studies will, however, be needed to determine whether otherandrogens can reproduce effects like those reported here for DHEA. Asthe concluding section, below, will suggest, the really importantquestion to be answered may, however, be even broader, and may, indeed,be a preview of the next big step forward in understanding ovarianphysiology.

D. New Concepts

A New Concept of Age-Related Declining Fecundity

IVF has revolutionized infertility care in many different ways. Amongstthe most significant are changes that go beyond medical considerationsand, indeed, may have societal impacts. For example, IVF gives us thetools to maximize pregnancy chances while minimizing multiple pregnancyrisks. It, however, has also revolutionized our clinical approachtowards women with significant degrees of DOR. Pregnancy and live birthrates in women at very advanced reproductive ages are better than ever,and women above age 40 represent now the proportionally most rapidlygrowing age group of U.S. women giving birth.

Young women, with normal age-appropriate OR, now conceive quickly withIVF.

Fertility centers, therefore, proportionally, now serve larger numbersof women with POA and/or DOR, as those accumulate disproportionally dueto their lower pregnancy chances. At our center, mean patient age in2009 has risen to approximately 39.5 years, and patients with prematureand age-dependent DOR represent close to 90 percent of all patients(FIG. 24: FIG. 24A is a graph showing trends in patient characteristicsof our center's IVF population—retrieval by year and age. Graph Ademonstrates mean ages for IVF patients between 2005 and year-to-date2009. FIG. 24B is a graph showing tends in patient characteristics ofour center's IVF population—percent retrievals by year and age. Graph Bdemonstrates the proportional shift from younger patients (<39 years) toolder women (≧40 years). FIG. 24C is a graph showing trends in patientcharacteristics of our center's IVF population—AMH by age category.Graph C demonstrates that this age shift is also accompanied by asignificant fall in AMH levels in younger women (ages 31-35 years) and,therefore, increasing DOR in these younger (POA) patients. Combined,these data explain why in 2009 close to 90% of the center's populationwas affected by either POA or DOR.). Our center's demographics may beextreme, and a biased reflection of the center's areas of specialclinical expertise and research interests. Infertility populations inall developed countries have, however, been graying.

The clinical problem of DOR has, therefore, progressively been movingfrom the periphery towards the center of priorities in infertility care.Effective clinical approaches towards DOR have, as a consequence,attracted considerable attention. We noted earlier that the indexpatient, aside from DHEA, found other potential remedies to enhanceoocytes yields in her literature search. Yet others have been addedsince and, even more importantly, a better understanding of underlyingpathophysiologies appears on the horizon.

Over the last few decades, and especially since IVF entered main streamclinical practice, ovarian stimulation protocols have been at theforefront of clinical research. Ovarian stimulation affects the ovary,however, only during approximately the last two weeks offolliculogenesis, when follicles become sensitive to gonadotropinstimulation. One of the reasons why, beyond their obvious clinicalsignificance, here summarized effects of DHEA supplementation arepotentially important, is their relevance for a much broaderunderstanding of ovarian physiology.

Present dogma suggests that women are born with their oocytes and thatthese oocytes age as women age. Our current understanding of decliningfemale fecundity with age, as previously noted, is based on decliningfollicle numbers and deteriorating egg quality with advancing femaleage. Here described DHEA work, however, raises serious questions aboutthe concept of declining egg quality with advancing ovarian age.

Young POA patients, while exhibiting other typical signs of ovarianaging, like elevated FSH, low AMH and ovarian resistance to stimulation,fail to demonstrate increased aneuploidy. Oocytes at that young age,thus, apparently are not yet functionally “old” enough to exhibit thedamage that would lead to aneuploidy. As noted earlier, oocytes in olderwomen do, leading to the well recognized increase in aneuploidy andmiscarriage rates with advancing female age.

As here summarized, DHEA supplementation apparently reduces theseeffects of age. This is best demonstrated by concentrating on availablemiscarriage data, which strongly suggest that DHEA supplementation inwomen of all ages significantly reduces miscarriage risk, andprogressively more effectively so, with advancing female age. Wepreviously already noted that the large decline in observed miscarriagescan statistically not be achieved without reduction in aneuploidy, andthat spontaneous pregnancy loss in very high risk populations formiscarriages is reduced to rates of normal fertile populations. Indeed,women with extremely low ovarian reserve, if they conceive, appear tohave the lowest miscarriage rates (FIG. 22).

In absence of normal oocytes, such low miscarriage rates areinconceivable. Whatever effects DHEA, therefore, may exert, they eitherhave to be able to revert “old” eggs into “young” ones (—a ratherunlikely option —) or one has to consider that oocytes, by themselves,really do not age.

Primordial oocytes, which make up the unrecruited egg pool thatconstitutes a woman's true OR, therefore, most likely do not really age,as current dogma holds. Indeed, they, likely, similar to cryopreservedgametes, hibernate at metabolic rates close to zero until recruited intofolliculogenesis. Once recruited, they pass the various stages offolliculogenesis within the age-dependent environment of a woman'sovary, which, of course, changes significantly as women age.

Under this concept it is not the egg that ages but the ovarianenvironment within which (—ever young—) oocytes mature throughfolliculogenesis. DHEA effects, under this concept, therefore, suddenlymake sense: DHEA supplementation into a (in comparison to younger age)deficient ovarian DHEA environments would be expected to causeenvironmental “rejuvenation,” and for upcoming cohorts of follicles toimprove folliculogenesis to levels usually found only in younger women.

Assuming this to be the correct concept, a revolution in treating DORappears possible, which will concentrate on maximizing conditions forall of folliculogenesis, rather than only its last two weeks ofgonadotropin sensitivity. Should progress be made in this direction, itseems likely that the female's reproductive lifespan can besignificantly extended. While egg numbers, of course, irreversiblydecline with advancing age, even menopausal ovaries still containfollicles and oocytes. In the Squirrel monkey, older animals,immediately prior to cessation of reproduction, still demonstrate anabundance of well-differentiated granulosa cells, Assuming thatunrecruited oocytes maintain their youth and that an aged ovarianenvironment can be rejuvenated, smaller, but healthier, egg cohorts mayallow for pregnancy into very advanced female ages.

DHEA, therefore, likely only represents a first drug in a whole newclass of pharmacological agents with potential to revert the ovarianenvironment to conditions, mimicking younger ages. Based on the knownloss of mitochondrial functions with advancing age, Bentov et al, forexample, recently suggested the use of mitochondrial nutrients, likecoenzyme Q10 (CoQ10), in women with ovarian senescence afterdemonstrating that CoQ10 increases oocytes numbers in older mice. On aside note, androgens positively affect mitochondrial function.

We, therefore, see DHEA as a forerunner for many new drug-developments,with the goal of recreating an ovarian environment in DOR patients,mimicking that of younger women. To get to this point, it will becomenecessary to determine the key differences between younger and olderovaries, which make the latter a relatively inhospitable environment forfolliculogenesis. Good potential technology for such studies hasrecently been developed.

Utilization Outside of Infertility

Above discussed findings and principles have so far been exclusivelypresented within the context of established infertility. Ovarian aging,however, does not only affect the infertile women. Indeed, the conceptof age-dependent fecundity is driven by the recognition that ovaries agein normally fertile populations with expected age-dependent adverseconsequences. These include progressively longer times to conception,increased aneuploidy and increased miscarriage risks, in principlesimilar to infertility patients.

One, therefore, can conceive of similar use of DHEA (and otherpharmaceutical compounds) in normal, fertile populations, attempting toconceive. Like folic acid, in attempts to prevent neural tube defects,supplementation of healthy individuals above certain ages, attempting toconceive, may have similar favorable public health consequences.

XIV. Additional Information on a New Evolving Concept of Ovarian Aging

Under currently widely held dogma, women are born with a pool ofprimordial follicles. As women age, eggs in these “stored” follicles agein parallel. Older women, therefore, have “poorer quality” eggs, whichlead to fewer pregnancies and more miscarriages. Summarizing currentunderstanding of ovarian aging, one, therefore, can say “it's the eggs,stupid!”

But, if that is really the case, and if eggs really age, how come womenwith poorest OR, if they conceive, produce such good embryos that hardlyany pregnancies are miscarried? It is this question that has preoccupiedus here at CHR for quite some months now and for which we, finally,believe to have found a reasonable answer. Moreover, should this answerturn out to be correct, then our understanding of ovarian aging needs tobe completely revamped.

Here is a short summary: We no longer believe that the eggs a woman isborn with age. Instead, we believe that these eggs are maintained in astate of, more less, physiologic suspension, akin to cryopreserved eggsor embryos, which, once frozen, remain in status quo. Once primordialfollicles are, however, recruited into folliculogenesis (a ca. 4.5months long process of follicular maturation), these very immature eggsare entering a journey towards maturation, which is depending on theovarian environment of the moment, which, of course, changes as womenage.

It, therefore, is not eggs that age, as women age, but the ovarianenvironment in which eggs mature.

This distinction is of crucial importance: It seems unlikely that agedeggs can be returned to youth. An aged environment, however, maypotentially be reversed with appropriate pharmaceutical therapies.Indeed, we now have come to believe that DHEA represents a first drugwhich has this kind of rejuvenating effect on the ovarian environment.By “correcting” the ovarian environment, DHEA allows for a follicularmaturation process in some patients that mimics that of younger women.As a consequence, egg and embryo quality is improved, pregnancy ratesare better and miscarriage rates are lower, as one would expect inyounger women.

Pharmacologic infertility treatments, practically since inception, haveconcentrated on the last two weeks of folliculogenesis, when folliclesbecome sensitive to gonadotropins. Under the here described new conceptof ovarian aging, DHEA, likely, represents the first of a new class offertility drugs, which, in contrast, affect folliculogenesis during thepreceding four months.

XV. Additional Details that DHEA Reduces Embryo Aneuploidy Based onDirect Evidence from Preimplantation Genetic Screening (PGS)

Dehydroepiandrosterone (DHEA) improves embryo quality and pregnancychances in women with diminished ovarian reserve (DOR) and may reduceaneuploidy. Since aneuploidy in human embryos is frequent and increaseswith advancing female age, a reduction in aneuploidy may explainimproved embryo quality and pregnancy rates.

Aneuploidy in preimplantation embryos can be demonstrated throughpreimplantation genetic screening (PGS). PGS is, however, only rarelyindicated in women with DOR, where often only small embryo numbers areavailable, and embryo selection, therefore, does not offer clinicalbenefits. PGS in such cases may, actually, reduce pregnancy chances within vitro fertilization (IVF).

DHEA supplemented patients have greater chances of at least one euploidembryo.

We investigate miscarriage rates after DHEA supplementation as asurrogate for aneuploidy risk. Since at least 60 percent of spontaneouspregnancy loss is attributable to chromosomal abnormalities, significantreductions in aneuploidy after DHEA supplementation may be reflected inlower miscarriage rates.

Results of this study strongly support the positive effect of DHEA onaneuploidy and PGS studies of human embryos offer direct evidence ofthis effect. See at least Example 11.

XVI. Improvement in Diminished Ovarian Reserve After DHEASupplementation

Ovarian reserve is defined by a woman's remaining follicular pool, whichdeclines with advancing female age. A patient is considered to sufferfrom diminished ovarian reserve if this pool, at any given age, issmaller than expected. Amidst considerable gains in the treatment ofinfertility, diminished ovarian reserve, whether due to physiologicalageing of the ovaries and premature ovarian aging, represent one of thefew unresolved problems of modern infertility care. Indeed, as treatmentsuccess with other infertility problems has improved, premature ovarianageing patients appear to increasingly aggregate in infertility centers,and women above age 40 years have become the proportionally most rapidlygrowing age group in US maternity wards, thus graying the populationunder infertility treatments.

DHEA is a mild, and therapeutically well tolerated, male hormone,produced as an intermediate step by adrenals (c. 85%) and ovaries (c.15%) during steroidogenesis, the conversion of cholesterol to the twosex hormones, oestradiol and testosterone.

Women with significant degrees of diminished ovarian reserve usuallyhave limited time left to conceive with use of autologous oocytes. SinceDHEA apparently increases oocyte yield, it also positively affectsovarian reserve. Ovarian reserve has traditionally been investigatedutilizing baseline FSH.

Anti-Müllerian hormone (AMH), produced by small post-primordial,pre-antral and antral follicles is a more specific reflection of ovarianreserve. Its utilization in association with prematurely diminishedovarian reserve has been advocated by different authors. We thereforeutilize AMH to assess ovarian reserve following DHEA supplementation inthis study.

DHEA supplementation significantly improves ovarian reserve in parallelwith longer DHEA use and is more pronounced in younger women. See atleast Example 12.

XVII. AMH Defines, Independent of Age, Low Versus Good Live-BirthChanges in Women with Severely Diminished Ovarian Reserve

Maximal receiver operating characteristic curve inflections, whichdifferentiate between better and poorer delivery chances in women withdiminished ovarian reserve (DOR) independent of age, were atanti-Müllerian hormone (AMH) 1.05 ng/mL (improved odds for live birth4.6 [2.3-9.1), 95% confidence interval; Wald 18.8, df=1], although livebirths occurred even with undetectable AMH. Pregnancy wastage was verylow at AMH≦0.04 ng/mL but significantly increased at AMH 0.41-1.05ng/mL, resulting in similarly low live-birth rates at all AMHlevels≦1.05 ng/mL and significantly improved live-birth rates atAMH≧1.06 ng/mL. See at least Example 13.

The following examples are to be construed as merely illustrative andnot limitative of the disclosure in any way.

EXAMPLE 1 Improved Ovulation

A study including a 43 year old woman, Patient A, undergoing IVF withbanking of multiple cryopreserved embryos for future aneuploidy screenand transfer is administered an androgen, namely DHEA. In ten months sheundergoes eight treatment stimulation cycles while continuouslyimproving her ovarian response, resulting in oocyte and embryo yieldsfar beyond those previously seen in a woman her age. Patient A's historyis unremarkable except for two previous malarial infections. She isallergic to sulfa medications and has a history of environmentalallergies. Her surgical history includes umbilical hernia repair at ageone and cholecystectomy at age 21. She had used oral contraceptives forover 10 years. She has no history of irregular menstrual cycles.

Day three serum FSH and estradiol (E2) in her first IVF cycle are 11mIU/ml and 18 pg/ml, respectively. In subsequent cycles her baseline FSHis as high as 15 mIU/ml. She is given an ovulation induction protocolwhich is prescribed for patients with evidence of decreased ovarianreserve. Briefly, the protocol includes the following medications:norethindrone acetate tablets (10 mg) for 10 days, starting on day twoof menses, followed three days later by a “microdose” dosage of 40 μg ofleuprolide acetate, twice daily, and, after another three days, by 600IU of FSH (Gonal-F; Ares-Serono, Geneva, Switzerland) daily. Peak serumE2 concentration on day nine of stimulation is 330 pg/ml. Followinginjection of 10,000 IU human chorionic gonadotropin (hCG), she undergoesoocyte retrieval. Only one oocyte is obtained and one embryo iscryopreserved.

Because of the poor response to ovulation stimulation, she is advised toconsider donor oocyte or embryo donation. She rejects both options. Shestarts a second cycle using the same stimulation protocol with oneexception: instead of 600 IU of FHS daily, Patient A received 450 IU ofFSH and 150 IU of human menopausal gonadotropin (HMG, Pergonal,Ares-Serono, Geneva, Switzerland). This stimulation protocol iscontinued in identical fashion for the remaining cycles. However, twoweeks before starting her second cycle, she begins administration of 75mg per day of oral micronized DHEA. The date on which she beginsadministration of 75 mg per day of oral micronized DHEA is Oct. 6, 2003.

Methods of Example 1

The eight treatment cycles are divided into three groups to allowstatistical comparison: pre-initiation and very early use of DHEA(early=cycles 1 and 2), initial cycles (mid=cycles 3-5), and latercycles (late=cycles 6-8). Comparison between these categories is byone-way analysis of variance (ANOVA) and multiple comparisons byStudent-Neuman-Keuls (SNK) test. The homogeneity of variances and usedorthogonal linear contrasts are tested to compare groups and polynomialcontrast to test for linear and quadratic trends. All outcomes arepresented as mean±1 standard deviation. Rate of change of oocyte counts,cryopreserved embryos and (log transformed) peak estradiol betweensubsequent cycles is estimated by linear regression.

Embryos are evaluated by the embryologists on day threepost-insemination for cell-count and grading. Embryo grading is based ona 1 to 4 scale depending on symmetry, percent fragmentation andappearance of the cytoplasm. All viable embryos are cryopreserved.Statistics are performed using SPSS for Windows, Standard version 10.0.7(SPSS Co., Chicago, Ill.). Assay of E2 and FSH are performed using theACS: 180 chemoluminescence system (Bayer Health Care LLC, Tarrytown,N.Y.).

A method of preconditioning ovulation induction in a human female isconceived, comprising administering an androgen in a female for at leastabout four consecutive months. In one embodiment, the androgen is DHEA.Administration of DHEA for at least about four consecutive months mayfurther comprise administering high dose gonadotropins to the female.Furthermore, DHEA may be administered along with follicle stimulatinghormone, human menopausal gonadotropin, norethindrone acetate,leuprolide acetate, and human chorionic gonadotropin. DHEA may beadministered orally.

The length of time the androgen is administered to the female can be atleast four consecutive months. The DHEA treatment may continue for morethan four months.

In one embodiment, the androgen administered is DHEA.

Results of Example 1

The results of ovulation induction are displayed in FIG. 1. After eightstimulation cycles and approximately eight months of DHEA treatment,Patient A produced 19 oocytes and 11 cryopreservable embryos. A total of50 viable embryos have so far been cryopreserved. Significantly moreoocytes (p=0.001) and cryopreserved embryos (p<0.001) are obtained inthe late cycles (cycles 6-8, 4+ consecutive months of DHEA treatment)compared to the combined early and mid cycles (cycles 1-5, 0-4consecutive months of DHEA treatment). There is no significantdifference in average embryo cell count (6.83±1.37 vs. 7.2±1.15) ormorphology (3.6±0.5 vs. 3.7±0.5) between early and mid compared to latecycles. Peak E2, total oocyte, and embryos cryopreserved increaselinearly from cycle to cycle, as shown in FIG. 1. Oocyte yield increase2.5±0.34 oocytes per cycle (p<0.001), cryopreservable embryo yieldincrease 1.4±0.14 embryos per cycle (p<0.001) and (log) peak E2 increase0.47±0.06 (p<0.001) across treatment cycles.

The linear increase in (log) peak E2 shown in FIG. 2 represents a cycleto cycle rate of increase from 123 pg/ml/cycle to 1491 pg/ml/cycle overthe eight cycles of treatment. After adjusting for cycle day, the(harmonic) mean E2 is 267 pg/ml (95% confidence intervals (CI) 143 to498 pg/ml) in the early phase, 941 pg/ml (95% CI 518 to 1712 pg/ml) inthe mid phase, and 1780 pg/ml (95% CI 1121 to 2827 pg/ml) in the latephase of treatment. Each of these homogeneous subsets is significantlydifferent from the other (p<0.05) by SNK multiple comparison testing.

The dramatic increase in oocyte and embryo yield experienced by this 43year old woman is completely surprising and unexpected. Patient A'spost-DHEA response to ovulation induction has become more like that of ayounger woman with PCO, than that of a 43 year old woman. Since startingDHEA treatment, Patient A has produced 49 embryos of high enough qualityto undergo cryopreservation. Sixty percent of those embryos wereproduced in the last three cycles of treatment, which took place afterat least about four consecutive months after starting treatment. Afterproducing only one embryo prior to starting DHEA treatment, Patient Aimproved by an order of magnitude and produced 13 oocytes and 9 embryosin a cycle after at least about four consecutive months of DHEAtreatment, 16 oocytes and 10 embryos in a cycle after at least aboutfive and a half consecutive months of DHEA treatment, and 19 oocytes and11 embryos in a cycle after at least about seven consecutive months ofDHEA treatment.

The increasing numbers of cryopreservable embryos due to DHEAsupplementation suggest improved embryo quality.

EXAMPLE 2 Improved Oocyte Fertilization and Cumulative Embryo Score

In another study, thirty (30) patients with evidence of decreasedovarian reserve were given supplemental DHEA 25 mg three times a day,for a total of 75 mg per day, for an average of about 4 months beforebeginning ovulation induction for IVF. Twelve patients contributed datafrom cycles both pre-DHEA and post-DHEA, eleven patients contributeddata from cycles only pre-DHEA, and seven patients contributed data fromcycles only post-DHEA. Patients' response to ovulation induction beforeDHEA treatment was compared to patients' response to ovulation inductionafter DHEA treatment with regard to peak estradiol, oocyte production,and embryos transferred and embryo quality.

The thirty patients contributed to data for 42 total cycles, 23 cyclesprior to and 19 cycles after starting DHEA supplementation. In comparingthe patients as a group pre- and post-DHEA treatment cycles, there wereimprovements in cancellation rate, peak estradiol, average day 3 embryocell counts, and embryo grade. However, average oocyte numbers, eggsfertilized, day-three embryos, embryos transferred and cumulative embryoscores increased significantly after DHEA treatment. In logisticregression models adjusted for oocyte number, there was evidence ofimproved fertilization rates (p<0.005), increased numbers of day-threeembryos (p<0.05), and of improved overall embryo score (p<0.01). In 34IVF cycles that reached the embryo transfer stage, a positive pregnancytest was obtained in zero of 16 cycles with less than an average ofabout 4 months of DHEA treatment and in 4/18 cycles after an average of4 months of DHEA treatment.

This case series illustrates that some ovarian function can be salvaged,even in women of advanced reproductive age.

TABLE 1 Univariate comparison of results of in vitro fertililizationbefore and after treatment with DHEA. Pre DHEA Post DHEA p N 23 19 Age40.9 ± 0.7  42.8 ± 0.7  ns Weeks of DHEA — 16.1 ± 2.4  — Cancellation5/21 (21%) 1/19 (5%) ns Peak Estradiol 1018 ± 160  1192 ± 904  nsOocytes 3.3 ± 0.7 5.8 ± 1.0 0.04  Fertilized eggs 1.3 ± 0.3 4.6 ± 0.8<0.001   Average Day 3 3.1 ± 0.6 4.5 ± 0.5 ns embryo cell count AverageDay 3 2.4 ± 0.3 2.8 ± 0.3 ns embryo grade Cumulative  34 ± 6.8  98 ±17.5 0.001 Embryo Score Transferred embryos 1.0 ± 0.2 2.6 ± 0.4 0.001Number of Day 3 0.9 ± 0.2 3.2 ± 0.6 0.001 embryos Positive hCG 0/16 4/18ns (per transfer cycle)

Cycle characteristics and responses to treatment are shown in Table 1.The average age of the patients who began DHEA was 41.6±0.6 years. Womenin the DHEA group used DHEA for a median value of 16 weeks before theirIVF cycle. The cycle cancellation rate was 5 of 21 cycles (21%) pre-DHEAand 1 of 19 (5%) post-DHEA. There was no statistically significantdifference in peak estradiol levels between pre- and post-DHEA cycles.

Continuing with the cycle outcomes presented in Table 1, there areimprovements in average cell count of day-three embryos and mean embryograde after DHEA treatment, however the differences are not significant.Mean oocyte numbers, fertilized eggs, day-three embryos, embryostransferred and cumulative embryo scores, all increased significantlyafter DHEA treatment. In the models adjusted for oocyte number, therewas still evidence of increased fertilization rates (1.93 fertilizedoocytes, 95% C.I. 0.82-3.04; p<0.005), increased numbers of day-threeembryos (1.36 embryos, 95% C.I. 0.34-2.4; p<0.05), and of improvedoverall embryo score (32.8, 95% C.I. 9.6-56; p<0.01).

FIG. 3 shows paired comparisons of fertilized oocytes (average increase2.5±0.60; p=0.002) among 12 patients with DHEA treatment cycles of lessthan about 4 weeks to fertilized oocytes in the same 12 patients afterat least about 4 weeks of DHEA treatment. FIG. 4 shows pairedcomparisons of day 3 embryos (average increase 2.0±0.57; p=0.005) among12 patients with DHEA treatment cycles of less than about 4 weeks and atleast about 4 weeks during IVF cycles. The paired comparisons shows thatthe mean increase in the number of fertilized oocytes was modest, butsignificant, (1.42±0.63 increased numbers of fertilized oocytes;p<0.05).

The mean increase in embryo scores was 57±14.7 (p<0.01). The increase inthe number of day 3 embryos was 2.0±0.57 (p=0.005) (See FIG. 4) and theincreased fertilization quantity was 2.5±0.60 fertilized oocytes perpatient (p=0.002) (See FIG. 3). DHEA supplementation improves theaverage oocyte numbers, eggs fertilized, day three embryos, embryostransferred, and cumulative embryo score.

In addition, DHEA supplementation also improves pregnancy rates anddecreases time to pregnancy. Two patients achieved ongoing pregnancieswhile taking DHEA without IVF; one (43 year old) while using DHEA duringa stimulated IUI (intrauterine insemination) cycle and a second (37 yearold) conceived spontaneously following an unsuccessful IVF cycle. Athird patient (40 year old) also conceived spontaneously while preparingfor an IVF cycle; however that pregnancy ended in a spontaneousabortion. In all 7 of 45 (16%) patients using DHEA have conceived and 5of 45 patients (11%) have experienced continuing pregnancies.

EXAMPLE 3 Increased Euploidy Rate

In another study (data not shown), patients were analyzed after fourweeks of DHEA treatment. Seven patients had embryos tested bypre-implantation genetic diagnosis (PGD). In three women who had PGDafter less than four weeks of DHEA usage and a mean age 41.5±5.1 at thetime of starting IVF cycles, the euploidy, or normal chromosome number,rate was 2/30 embryos (6.6%). In six patients who had PGD after morethan four weeks of DHEA usage, and a mean age of 43.7±1.3 years at thetime of starting IVF cycles, the euploidy rate increased to (8/27;29.6%), though this trend did not reach statistical significance. Thereis a mean age difference between patients who underwent IVF after lessthan four weeks of DHEA usage (mean age 41.5±5.1) and patients whounderwent IVF after at least four weeks of DHEA usage (mean age43.7±1.3).

As women age, there is a substantial decline in euploidy rates inembryos produced. Thus, the increase in euploidy results in older womenis dramatic evidence of the effectiveness of DHEA in improving embryoquality.

EXAMPLE 4 DHEA Treatment Increases Euploidy Number

In a series of studies, it has been documented that DHEA supplementationin women with diminished ovarian reserve (DOR) increases egg and embryocount, improves egg and embryo quality, increases pregnancy rates, andshortens time to conception.

The reports of the studies point towards improvements in follicularrecruitment after treatment with androgenic compounds. Since DHEAeffects are statistically significant after approximately four months,and since this time period is approximately reflective of the fullfollicular recruitment cycle, we concluded that DHEA may, at least inpart, affect follicular recruitment processes, possibly by influencingapoptosis. Androgens have been reported to affect granulosa cellapoptosis.

While women with prematurely DOR appear to have normal embryonicaneuploidy rates, older women, with physiologic aging ovaries,demonstrate very high aneuploidy rates of their embryos. Increasinganeuploidy rates with advancing female age are, therefore, considered aprimary cause for diminishing pregnancy chances, and an increasingmiscarriage risk, in older women. Since treatment with androgeniccompounds in such patients appears to improve embryo quality andpregnancy chances, it is likely that such treatment positively affectsaneuploidy rates.

Materials and Methods of Example 4

All the IVF cycles performed at the Center for Human Reproduction (CHR)in New York, N.Y., between 2004 and 2006 for cycles performed in womenwith a diagnosis of DOR were retroactively reviewed. The studypopulation, involving 27 IVF cycles, was selected amongst those cycleswhich, in addition, had undergone preimplantation genetic diagnosis(PGD).

The diagnosis of DOR was made based on previously reported abnormallyhigh, age stratified baseline FSH levels. In practical terms, this meantthat a diagnosis of DOR was reached if baseline FSH levels exceeded the95% confidence interval of age appropriate levels, independent of priorIVF retrievals and/or oocyte numbers. At, or above age 43, all patientswere considered to suffer from DOR, independent of baseline FSH level.

Since the year 2004, women with proven DOR, who had undergone at leastone prior ovarian stimulation, demonstrating ovarian resistance based oninadequately low oocyte numbers, routinely were offered oral DHEAsupplementation (25 mg TID) prior to any further IVF cycle starts. Ifunder age 40, DHEA was given for up to four months prior to IVF. Womenof older age received DHEA, if possible, for at least two months.

Women with DOR, who had no proof of ovarian resistance, were not placedon DHEA supplementation until such proof was obtained, unless they wereat, or above, age 43 years. IVF cycles on DHEA supplementation have,therefore, to be considered as more severely affected by DOR than thosecycles that were conducted without such supplementation. This fact isalso reflected by the baseline cycle characteristics of DHEA-treated,and -untreated, patients (Table 2), which demonstrate trends towardsolder age and higher baseline FSH levels in DHEA treated patients.

TABLE 2 Baseline characteristics of DHEA-treated, and -untreated,patients¹ DHEA -TREATED DHEA - UNTREATED n = 8 n = 19 Age (±SD, year)41.2 ± 4.7 38.9 ± 5.1  Baseline FSH² ± SD 12.4 ± 9.2 9.0 ± 2.7 (mIU/ml)Baseline Estradiol² ± SD  59.7 ± 32.2 68.1 ± 59.1 (pg/ml) ¹None of thebaseline parameters, listed in the table, differed to a statisticallysignificant degree between the two groups. ²Reflects highest baselinelevel of each patient, and not necessarily the baseline level of the IVFcycle.

For the purpose of this analysis, a patient had to be for at least onemonth (30 days) on DHEA supplementation in order for the IVF cycle to beconsidered amongst DHEA-treated cycles. All other DOR patients wereconsidered to have received no DHEA treatment. Following thisdefinition, 19 DOR patients had received no DHEA supplementation, andeight had.

All women with DOR, independent of DHEA supplementation, were stimulatedwith identical protocols, as previously reported in detail elsewhere. Inshort, they, without exception, received a microdose agonist protocolwith maximal goandotropin stimulation of 600 IU to 750 IU daily, withpreponderance of FSH, and a smaller daily amount of human menopausalgonadotropin (hMG).

PGD was performed in routine fashion, as also previously described indetail, and involved the analysis of chromosomes X, Y, 13, 16, 18, 21and 22 by fluorescence in situ hybridization (FISH) on day three afterfertilization. Embryo transfer occurred on day five after fertilization.

Patients were represented by only one cycle outcome in each group. Ifpatients had undergone more than one cycle, either with, or without,DHEA supplementation, only their latest cycle was included in theanalysis. Three patients underwent both a pre-DHEA and a post-DHEA cycleand in those cases both cycles were included in the analysis.

Statistical analysis was performed using SPSS for windows, standardversion 10.0.7. Data are presented as mean±one standard deviation,unless otherwise noted, and statistical differences between the twostudy groups were tested by Chi-square and (two-sided) Fisher's ExactTest, where applicable, with significance being defined as p<0.05.

Results of Example 4

A total of 27 consecutive IVF cycles in women with DOR who also hadundergone preimplantation genetic diagnosis (PGD) were identified andevaluated. Amongst those, 19 had entered IVF without DHEA treatment and8 had received DHEA supplementation for at least four weeks prior to IVFstart.

Table 3 summarizes cycle outcomes.

TABLE 3 IVF cycle and PGD outcomes DHEA - TREATED DHEA - UNTREATED PeakEstradiol ± SD 2310.3 ± 1108.1 2123.3 ± 1054.7 (pg/ml) Oocytes ± SD 10.4± 7.3  8.5 ± 4.6 Embryos ± SD¹ 9.1 ± 7.3 5.7 ± 2.7 n Euploid ± SD 2.1 ±1.4 1.6 ± 2.3 % Euploid ± SD 44.1 ± 37.8 21.4 ± 27.5 n Aneuploid ± SD4.4 ± 3.0 3.5 ± 0.3 % Aneuploid ± SD 55.9 ± 37.8 78.6 ± 27.5 Patientswith euploid 8/8 (100)² 7/13 (53.8)² embryos (%) SD, standard deviationof mean; ¹Reflects total number of embryos. Since only high quality6-cell to 8-cell day-3 embryos undergo PGD, the number of embryos testedfor ploidy was smaller. ²Reflects a statistically significant differenceby Likelihood ratio (p = 0.004) and (two-sided) Fisher's Exact Test; p =0.026. Other comparisons in this table did not reach statisticalsignificance.

DHEA treatment resulted in trends towards higher oocyte numbers(10.4±7.3 vs. 8.5±4.6). A significantly larger number of DHEA treatedIVF cycles (eight out of eight, 100%) had at least one euploid embryofor transfer than in untreated cycles (10/19, 52.6%; Likelihood ratio,p=0.004; Fisher's Exact Test, p=0.026). In other words, the primaryresult reaching statistical significance was the difference in thepercentage of IVF cycles which resulted in the transfer of at least oneeuploid embryo, with DHEA treated patients reaching embryo transfer in100 percent of cycles, while untreated patients did so in only 52.6percent of cases.

As can be seen in Table 3, peak estradiol levels, oocyte and embryonumbers and the results of PGD, all demonstrated trends towards abeneficial effect of DHEA. Peak estradiol levels were higher and oocyte,as well as embryo numbers, were larger. There was also a trend towardsmore euploidy in embryos from treated cycles, both in absolute numbersand in percentages of embryos evaluated by PGD.

Amongst the 27 reported cycles, three patients contributed pre- andpost-DHEA cycles. When these cycles were separately analyzed, theydemonstrated similar trends as observed for the whole study (Table 4).

TABLE 4 IVF cycle parameters in 3 women with DHEA and -no-DHEA cycles¹Age ± SD (years) 38.2 ± 5.5 Baseline FSH² ± SD (mIU/ml) 10.5 ± 1.5Baseline Estradiol² ± SD (pg/ml)  54.4 ± 21.7 DHEA - TREATED DHEA -UNTREATED Time pre-/post DHEA 2.4 ± 2.5 1.9 ± 2.2 (months) Oocytes ± SD6.0 ± 4.8 4.8 ± 1.0 Total Embryos ± SD 4.0 ± 2.7 4.5 ± 0.6 AneuploidEmbryos 2.0 ± 1.8 3.5 ± 0.6 SD, standard deviation; ¹None of thedifferences between the two study groups reached statisticalsignificance, ²Reflects highest baseline level of patients, but notnecessarily baseline level during IVF cycle.Discussion of Example 4

The here presented study demonstrates evidence that DHEA improves, to astatistically significant degree, the number of euploid embryosavailable for embryo transfer after IVF, and may be at least a partialexplanation of why DHEA supplementation improves pregnancy chances inwomen with DOR. The study also demonstrates a trend towards higherpercentages of euploid embryos after DHEA and higher absolute numbers ofeuploid embryos. The here observed effect of statistically moretransferable, euploid embryos, may be due to larger oocyte and embryonumbers, lower aneuploidy rates, or both effects combined.

The mean number of euploid embryos increased after DHEA treatment byapproximately one-half embryo. One-half additional embryo, especially ifproven euploid, represents significant additional pregnancy potential inwomen with DOR, who usually produce only relative small embryo numbers.Indeed, this reflects approximately a one-third improvement in euploidembryo yield, and results in the availability of at least one embryo fortransfer in all post-DHEA cycles. In comparison, only 52.6% of untreatedcycles achieved the same goal. This is a statistically significantdifference in embryo transfers. Pretreatment with DHEA of women with DORsignificantly increases their chances for the transfer of at least oneeuploid embryo and may, therefore, at least in part, explain the higherpregnancy rates reported with DHEA supplementation.

Based on the incremental improvement in DHEA effects for up to fourmonths, and the correlation of the time span to a full cycle offollicular recruitment, it is suspect that DHEA may affect apoptoticprocesses during follicular recruitment. As a consequence, more healthyfollicles survive maturation, reach the stage of gonadotropinsensitivity and become subject to exogenous gonadotropin stimulation.These, in turn, also could be expected to have a higher probability ofeuploidy.

Increasing aneuploidy rates with female age are considered the principlecause of decreasing spontaneous female fertility, increasing infertilityand rising miscarriage rates. DHEA may improve euploidy rates as will bediscussed in more detail herein, and in turn, improves spontaneousfemale fertility, decreases the rate of female infertility and reducesmiscarriage rates.

EXAMPLE 5 DHEA Substitution Improves Ovarian Function

In a further study, a case of probable 17, 20-desmolase deficiency,resulting in abnormally low estradiol, DHEA, androstenedione andtestosterone levels, is presented in a woman with a clinical history of,initially, unexplained infertility and, later, prematurely agingovaries.

This patient started attempting conception in 1996, at age 33. Afterfailing to conceive for over one year, she was diagnosed withhypothyroidism and was placed on levoxyl. She, thereafter, remainedeuthyroid for the whole period described in this case report. Sheentered fertility treatment at a prominent medical school based programin Chicago, in August of 1997, where, now age 34, she failed threeclomiphene citrate cycles. No further treatment took place until alaparoscopy was performed in October of 1999, at a prominentAtlanta-based infertility center (where the couple had relocated to),revealing stage II endometriosis which was laser vaporized. Followingsurgery, a fourth clomiphene citrate cycle and a firstgonadotropin-stimulated cycle failed. Table 5 presents selected key labdata for all ovarian stimulation cycles the patient underwent. A firstin vitro fertilization (IVF) cycle was performed, at age 36, in Octoberof 2000.

This cycle resulted in expected oocyte and embryos yields. Three embryoswere transferred, resulting in a chemical pregnancy. Three other embryoswere cryopreserved. However, because of a persistently thin endometrium,a number of attempts at transfer were cancelled.

In April of 2001, the patient was, based on an abnormal glucosetolerance test, diagnosed with insulin resistance, and was placed onmetformin, 500 mg thrice daily. She had no signs of polycystic ovariandisease: her ovaries did not look polycystic, she was not overweight,had no signs of hirsutism or acne, and androgen, as well as estradiol,levels were in a low normal range (Table 2). In June of 2001 (age 37), asecond IVF cycle was initiated. In this cycle the patient demonstratedthe first evidence of ovarian resistance to stimulation in that sheproduced only six oocytes. Only one out of five mature oocytefertilized, despite the utilization of intracytoplasmic sperm injection(ICSI). The previously cryopreserved embryos were, therefore, thawed andtransferred, together with the one fresh embryo from the current cycle.The transfer was unsuccessful.

In August of 2001, the female's FSH level for the first time wasabnormally elevated (11.4 mIU/ml), with estradiol levels remaininglow-normal. Subsequent FSH levels were 19.1, 9.7 and 9.8 mIU/ml inNovember and December (twice), respectively, all with low-normalestradiol levels. FSH levels continued to fluctuate in 2002, with levelsreported as 11.4 mIUI/ml in February, 8.7 in March, 13.6 in June and19.6 in September, while estradiol levels remained persistentlylow-normal (Table 2).

A third IVF cycle was started in October of 2002, with a baseline FSH of11.3 mIUI. Ovarian stimulation, which in the prior two cycles had beengiven with only recombinant FSH (and antagonists), was now given in acombination of recombinant FSH and hMG at a combined dosage of 300 IUdaily. Estradiol levels reached only 890 pg/ml and only 5 oocytes wereretrieved. All four mature oocytes fertilized and four embryos weretransferred. A twin pregnancy was established by ultrasound and asingleton by heart beat. This pregnancy was, however, miscarried andconfirmed as aneuploid with a Trisomy 22.

The fact that this cycle, after the addition of hMG to the stimulationprotocol, appeared more successful, led the patient to a search of themedical literature. Like our previously reported patient (Barad andGleicher, 2005), this patient discovered a case series. The paperattracted the patient's interest. In follow up, she asked a medicalendocrinologist to evaluate her adrenal function. An initial evaluationrevealed very low DHEA, DHEA-S, androstenedione and testosterone levels(Table 2). An ACTH-stimulation test was ordered which showed theexpected increase in cortisol level, but unchanged, low DHEA, DHEA-S andtestosterone levels (Table 3). The patient was advised by her medicalendocrinologist that the most likely explanation for such a finding wasa 3-beta hydroxysteroid dehydrogenase deficiency. This enzyme defect is,however, associated with an accumulation of DHEA and, therefore, highlevels of the hormone. (Speroff et al., 1999a). Such a diagnosis for thepatients is, therefore, unlikely. Instead, abnormal 17,20-desmolase(P450c17) function would be expected to result in exactly the kind ofhormone profile, reported in this patient after ACTH stimulation,characterized by persistently low DHEA, androstenedione, testosteroneand estradiol levels, but normal aldosterone and cortisol levels.

In July of 2003, the patient was started on 25 mg daily of micronizedDHEA. After five weeks of treatment, DHEA, DHEA-S and androstenedionelevels had normalized into mid-ranges. (Even though androstenedione ispartially produced through the activity of 17,20-desmolase from17-hydroxyprogesterone, part is also derived from DHEA through theactivity of 3-beta hydroxysteroid dehydrogenase [Speroff et al., 1999a].The normalization of androstenedione, after DHEA administration,therefore, also speaks for an underlying 17,20-desmolase defect, and nota 3-beta hydroxysteroid dehydrogenase deficiency.) In the third andfourth month, following the start of DHEA supplementation, the patientovulated spontaneously with estradiol levels of 268 and 223 pg/ml (Table2), respectively, measured on the day of LH surge.

On Jan. 28, 2004 (age 39), and after DHEA therapy of approximately sixmonths, a fourth IVF cycle was initiated. Her baseline FSH level in thatcycle was 9.6 mIU/ml, estradiol 56 pg/ml. Stimulation took place with300 IU of recombinant FSH (without hMG) and with an agonist flareprotocol. Estradiol levels reached a peak of 1764 pg/ml, 8 oocytes wereretrieved, six out of seven mature oocytes fertilized and six embryoswere transferred. A triplet pregnancy was established with heart beats.Two, out of the three fetuses lost heart beat spontaneously, and thepatient delivered by cesarean section, at term, a healthy singleton maleinfant.

At surgery, her ovaries were closely inspected and described as “old”and “small”, with the left one being described as “almost dead.” DHEAand DHEA-S levels at six months of pregnancy were reported at “recordlows.” DHEA-S, six weeks post-delivery, was still very low (Table 5). Attime of this report, the male offspring is nine months old and themother has been re-started on DHEA in an attempt at another pregnancy.

DHEA substitution resulted in apparently normal peripheral DHEA levels,spontaneous ovulation and normal estradiol production by the ovaries. AnIVF cycle, after approximately six months of DHEA substitution, showed,in comparison to a pre-DHEA IVF cycle, improved peak estradiol levels,increased oocyte and embryo numbers and resulted, at age 39, after 6years of infertility therapy, in a triplet pregnancy and a normalsingleton delivery.

Low DHEA levels appear associated with female infertility and ovarianaging. DHEA substitution normalizes peripheral DHEA levels and appearsto improve ovarian response parameters to stimulation.

The reported patient exhibited some of the classical signs ofprematurely aging ovaries (Nikolaou and Templeton, 2003; Gleicher N,2004) which include ovarian resistance to stimulation, poor egg andembryo quality and prematurely elevated FSH levels. The patient wasinitially thought to have largely unexplained infertility. She laterdeveloped quite obvious signs of prematurely aging ovaries and, finally,even showed elevated FSH levels.

It has been previously suggested that the decrease in DHEA levels, withadvancing female age, may be an inherent part of the ovarian agingprocess and may, at least in part, and on a temporary basis, be reversedby external DHEA substitution (Barad and Gleicher, 2005, 2005a). Thiscase demonstrates that low DHEA levels are, indeed, associated with allthe classical signs of both prematurely and normally aging ovaries.While association does not necessarily suggest causation, the observedsequence of events in this patient supports the notion that low DHEAlevels adversely affect ovarian function.

Once the patient was administered oral DHEA, a reversal of many findingscharacteristic of the aging ovary, were noted. First, the patient's DHEAand DHEA-S levels normalized. In subsequent natural cycles an apparentlynormal spontaneous follicular response was observed, with normalovulatory estradiol levels in a patient with persistently low estradiollevels before DHEA treatment (Table 5). The response to ovarianstimulation improved, quantitatively and qualitatively, as the patientimproved peak estradiol levels, oocyte and embryo numbers and, as thesuccessful pregnancy may suggest, also embryo quality.

One cannot preclude that other factors contributed. For example, theovarian stimulation protocol had switched from an antagonist to anagonist flare protocol. The data demonstrates that a maximal effect ofDHEA is achieved after at least about four consecutive months of use.This patient was on DHEA treatment for approximately six months beforeshe conceived the pregnancy that led to her first live birth.

This case is well documented in its DHEA deficiency and in its mostlikely cause. The reported adrenal response to ACTH stimulation (Table5) lends itself to the explanation (FIG. 1) of 17,20-desmolasedeficiency.

TABLE 5 Relevant laboratory results Date TEST RESULT (Normal values)*COMMENTS August 1997 TSH 7.8 mlU/l (0.4-5.5) Diagnosis of hypothyroidismMay 1999 FSH 4.0 mIU/ml April 2001 Glucose tolerance test Elevated ½hour insulin levels Diagnosis of Normal Glucose levels insulinresistance June 2001 FSH 7.7 mIU/ml Estradiol 33 pg/ml August 2001Testosterone 2 ng/dl  (3-29) free/weakly bound free only 1 pg/ml  (1-21)total 13 ng/dl (15-70) DHEA-S 96 mcg/dl  (12-379) Total Cortisol 14.2mcg/ml  (4-22) FSH 11.4 mIU/ml Diagnosis of prem. ov. aging Estradiol 45pg/ml October 2001 Estradiol periovulatory 119 pg/ml November 2001Testosterone total 23 ng/ml (14-76) Androstenedione 98 ng/ml  (65-270)Ovarian antibodies negative FSH 19.1 mIU/ml Estradiol 23 pg/ml December2001 FSH 9.7 mIU/ml Estradiol 27 pg/ml February 2002 Testosterone total<20 ng/dl (20-76) Androstenedione 76 ng/dl  (65-270) FSH 11.4 mIU/mlEstradiol 28 pg.ml March 2002 Testosterone total 16 ng/dl (15-70) FSH8.7 mIU/ml Estradiol 29 pg/ml May 2002 FSH 13.6 mIU/ml Estradiol 30pg/ml periovulatory 139 pg/ml June 2002 periovulatory 50 pg/ml September2002 Testosterone total 15 ng.dl (15-70) free 1.6 pg/ml  (1-8.5) % free0.0107 (0.5-1.8) Estradiol periovulatory 136 pg/ml October 2002 FSH 11.3mIUI/ml Estradiol 43 pg/ml February 2003 FSH 13.6 mIU/ml Estradiol 33pg/ml March 2003 FSH 8.9 mIU/ml Estradiol 67 pg/ml May 2003 Anti-adrenalantibodies negative Estradiol periovulatory 139 pg/ml DHEA 132 ng/dl(130-980) DHEA-S 79 mcg/dl  (52-400) Testosterone total 34 ng/dl (20-76)free 3 pg/ml  (1-21) July 2003 DHEA TREATMENT START DHEA 296 ng/dl(130-980) DHEA-S 366 mcg/dl (52-400) Androstenedione 121 ng/dl (65-270)September 2003 Estradiol periovulatory 268 pg/ml October 2003 FSH 14.7mIUI/ml Estradiol 44 pg/ml periovulatory 224 pg/ml November 2003 FSH 17mIU/ml Estradiol 38 pg/ml December 2003 DHEA 278 ng/ml (130-980) DHEA-S270 mcg/dl  (52-400) Testosterone total 25 ng/ml (20-76) free and weeklybound 4 ng/dl  (3-29) free 2 pg/ml  (1-21) January 2004 FSH 18 mIU/mlFSH 9.6 mIU/ml 4^(th) IVF Estradiol 56 pg/ml CYCLE START August 2004MID_PREGNANCY DHEA 74 ng/dl (135-810) DHEA-S 27 mcg/dl (**) October 2004DELIVERY December 2004 DHEA-S 52 mcg/dl  (44-352) *Laboratory tests wereperformed at varying laboratories (**) No Pregnancy levels availablefrom laboratory

TABLE 6 ACTH stimulation test HORMONE BASELINE +30 MINUTES +60 MINUTESDHEA-S (mcg/ml) 87 88 83 Cortisol total (mcg/dl) 15 26 27 Testosteronetotal (ng/dl) 28 32 33 free and weakly bound 5 5 5 free 3 3 3

This case report presents further evidence for DHEA deficiency as acause of female infertility and as a possible causative agent in theaging processes of the ovary. It also presents further confirmation ofthe value of DHEA substitution whenever the suspicion exists thatovaries may be lacking of DHEA substrate. Finally, this case reportraises the important question what the incidence of adrenal17,20-desmolase (P450c17) deficiency is in women with prematurely agingovaries.

EXAMPLE 6 Increase Male Fetus Sex Ratio

Androgenization of females with dehydroepiandrosterone (DHEA), as werecently have been utilizing in the fertility treatment of women withdiminished ovarian reserve, in combination with the investigation ofspontaneous, versus in vitro fertilization (IVF)—conceived, pregnanciesallows for an investigation of the basic theory of sex allocation andits possible pathophysiologic mechanisms.

The treatment protocol for long-term supplementation with DHEA that mayimprove oocyte and embryo quantity, quality, pregnancy rates and time toconception in women with diminished ovarian reserve, involves 25 mg ofmicronized, pharmaceutical grade DHEA, TID will usually uniformly raiselevels of unconjugated DHEA above about 350 ng/dl, and, therefore, raisebaseline testosterone. Estradiol baseline levels may also be raised.

A retroactive review of either ongoing or delivered pregnancies beyond20 weeks gestational age, conceived while on DHEA treatment for at least60 days, revealed 23 women. A total of 19 pregnancies were recorded with16 singleton and 3 twin pregnancies. The medical records of all 19 womenwere reviewed in order to determine whether they conceivedspontaneously, defined as including pregnancies conceived withintrauterine inseminations, or by IVF. If conception had occurred byIVF, it was recorded whether fertilization was spontaneous or byintracytoplasmic sperm injection (ICSI).

As a control group, seven women were selected who had undergone one IVFcycle with preimplantation genetic diagnosis (PGD), while for at least60 days on DHEA supplementation, but had not conceived. The PGD data,defining each embryo's gender, were recorded. Statistics were performedusing a binomial runs test, comparing seen distributions with anexpected distribution of 50 percent, with p≦0.05 defining significance.

Sixteen singleton pregnancies resulted in 11 males and 5 females (N.S.).Two of three twin pregnancies were heterozygous and one homozygous. Ifoutcomes of both heterozygous twins, but of only one homozygous twin,were added, the final gender distribution was 15 males and 6 females(p=0.078, N.S.)

Amongst six pregnancies, spontaneously conceived, the distributionbetween female and male offspring was equal, at three and three,respectively. Whereas amongst the remaining 15 offspring, which wereproducts of pregnancies achieved through IVF, the distribution was 12males and 3 females (p=0.035). Only one IVF patient failed to have ICSI.Amongst women undergoing IVF and PGD, 53 embryos were analyzed from 17IVF cycles, all having undergone ICSI. The gender distribution was notsignificantly skewed, with 27 being male and 26 female.

This study allows for the dissection of the conception process into itsvarious stages and, therefore, permits an analysis of, not only thebasic question whether androgenization does indeed, affect genderselection in the human, but also how such a selection may be influenced.

The here presented data, demonstrating a strong trend towardssignificance overall, and significance (p=0.035) amongst IVF patients,suggest, convincingly that gender determination may be influenced byhormonal environment. Assuming an effect of androgens on genderselection, such women should give birth to a preponderance of maleoffspring. Confirming such a finding could present a potentialadditional explanation for the evolutionary preservation of PCOS inpractically all human races.

EXAMPLE 7 Increase Pregnancy Rates

In an additional study, a retrospective analysis of a 190 women withdiminished ovarian function above 30 years old, who were treated between1999 and December 2005 was completed to assess the impact of DHEAsupplementation on the time interval to the establishment of pregnancy.

A prequalification for each patient's diagnosis of diminished ovarianfunction was either a sub-diagnosis of premature ovarian aging (POA) ora sub-diagnosis of diminished ovarian reserve (DOR). POA was, in turn,defined as baseline follicle stimulating hormone (b-FSH), on Day 2/3 ofa cycle as <12 mIU/ml, but exceeding the 95% CI of the mean value forthe patient's age group.

Specifically, this meant b-FSH≧7.4 mIU/ml at age 30-34 years and ≧8.6mIU/ml at age≧35 years. DOR, in turn, was defined as b-FSH≧12 mIU/mland/or a baseline estradiol level≧75 pg/ml. 49 patients were confirmedwith POA and 52 patients were confirmed with DOR, creating a controlgroup of 101 women. Because of potential impending loss of ovarianfunction in the control group women, the control group women weretreated with IVF as soon as possible.

During the time studied, the study group consisted of 89 patients, withdiminished ovarian function (POA 24, DOR 65). Each person in the studygroup was placed on DHEA supplementation. The DHEA supplementationincluded administering about 25 mg of (pharmaceutical grade) micronizedDHEA, three times daily, for up to about four months (mean 3.8±0.3months). In contrast to the control group, women in the study group didnot enter IVF right away. This delay of IVF treatment allowed thepossibility of spontaneously conceived pregnancies. Those patients whodid not conceive spontaneously within four months of beginning DHEAentered IVF.

Methods of Example 7

Ovarian stimulation was identical for study and control groups andcomprised microdose agonist flare, followed by maximal dosagegonadotropin stimulation, using about 300-450 IU of FSH and about 150 IUof HMG. Study patients received DHEA continuously until a positivepregnancy test was obtained or until the patient dropped out oftreatment. DHEA and DHEAS levels were monitored monthly, and patientswere interviewed at each visit about adverse reactions to DHEAsupplementation. Because of the dynamics of the DHEA treatmentalgorithm, at the time of this data analysis, 16 women in the studygroup were at various stages of DHEA supplementation, prior to anyintervention, 9 women received ovarian stimulation while on DHEA, and 64have undergone an IVF cycle.

In order to assess the impact of DHEA supplementation on time intervalto the establishment of pregnancy, this study was designed as alife-table analysis, measuring not only total pregnancy rates but alsothe time between initial presentation and end of last treatmentintervention.

Each recorded clinical pregnancy, defined as positive fetal cardiacactivity on ultrasound examination after 6 weeks, was recorded as apositive outcome. Patients who continued treatments beyond the studyperiod or stopped treatments were considered right censored data at theend of the study period, or at treatment cessation, respectively.

The following factors were compared between study and control groups:female age, months of infertility prior to initial visit, length oftreatment from first presentation, gravidity, race, IVF treatments,maximal baseline FSH levels, maximal baseline estradiol levels, IVFcycle cancellation rates, oocyte numbers, number of embryos transferred,implantation rates, cumulative clinical pregnancy rates and miscarriagerates.

A Cox regression analysis was used to evaluate time-to-event. The modelthat we used stratified for level of ovarian reserve (POA and DOR) andadjusted for age, day 3 FSH, fertility treatments (none, IntrauterineInsemination and controlled ovarian hyperstimulation (IUI/COH), or IVF)and race/ethnicity. A trend in pregnancy rates over months of DHEAexposure with an interaction term for time and DHEA months of exposurewas tested. SPSS for Windows, Standard version 10.0.7 (SPSS Co. Chicago,Ill.) was utilized for data analysis. Continuous outcomes are presentedas mean±1 standard error. Univariate comparisons were made with analysisof variance, or by using Fisher's exact test.

Results of Example 7

Table 7 summarizes patient characteristics. As can be seen, studypatients were slightly older than the controls at 41.6±0.4 and 40.0±0.4years (p<0.05) respectively. Pregnancy histories, duration ofinfertility and of treatment (in months) were similar between the twogroups. Controls represented a non significant larger proportion ofminorities, received more treatment cycles overall (1.6±0.9 versus1.3±1.0; p<0.05) and differed significantly in the various treatmentsthey received (p<0.001). Study patients demonstrated a non-significantlyhigher b-FSH 16.0±1.2 13.6±1.0 mIU/ml) and a significantly higherbaseline estradiol level (366±53 versus 188±24 pml/ml; p<0.05). Morewomen in the study group had b-FSH≧10 mIU/ml that amongst controls (73%versus 51.5%; p<0.05). In addition, greater proportion of women in thestudy group had DOR (p<0.005).

TABLE 7 Characteristics of DHEA Treated and Controls DHEA Control p N 89101 Age 41.6 ± 0.4 40.0 ± 0.4 <0.05 Months Infertility 44.5 ± 4.8 41.9 ±6.9 ns Months from First Visit  8.1 ± 0.7  7.8 ± 1.0 ns Race ns White 62(70.5%) 57 (56.4%) — Hispanic 7 (7.9%) 12 (11.9%) — Black 9 (10.2%) 14(13.9%) — Asian 11 (12.5%) 18 (17.8%) — Cycles of Treatment 1.3 ± 1  1.6 ± 0.9 <0.05 Treatment <0.01 No Treatment 16 (18.2%) 0 (0%) —IUI/COH 9 (10.2%) 0 (0%) — IVF 64 (71.6%) 101 (100%) — Day 3 FSH(mIU/ml) 16.0 ± 1.2 13.6 ± 1.0 ns Day 3 E2 (pmol/ml) 366 ± 53 188 ± 24<0.05 Ovarian Function  <0.005 POA 24 (27%) 49 (48.5%) — DOR 65 (73%) 52(51.5%) —

Table 8 lists the results of univariate comparisons of treatmentoutcomes. As can be seen, confirming a more severe degree of diminishedovarian function, the study group produced significantly fewer oocytes,normal day-3 embryos (2.4±0.03 versus 3.5±0.2; p<0.05) and transferredembryos (2.1±0.2 versus 2.7±0.2; p<0.05). Cycle cancellations were,however, nominally higher among the controls (25.7% versus 14.3%).

TABLE 8 Univariate Comparison of Results Between Control and DHEATreated Patients DHEA Control p N total; (IVF) 89 (64) 101 Months ofDHEA 3.8 ± 0.3 — — Cancellation (IVF) 9/63 (14.3%) 26/101 (25.7%) nsOocytes 3.9 ± 0.4 5.8 ± 0.5 <0.01 Normal Day 3 embryos 2.4 ± 0.3 3.5 ±0.2 <0.05 Transferred embryos 2.1 ± 0.2 2.7 ± 0.2 <0.05 Positive hCG(>25 mIU/ml) 26/88 (30%) 18/101 (18%) ns Implantation (FH/Embryos 13/101(11.4%) 11/148 (6.9%) ns trans) Clinical Pregnancy 25/89 (28.1%) 11/101(10.9%) <0.01 No Treatment 6/16 (35.3%) — — IUI/COH 6/9 (66.7%) — — IVF13/64 (20.6%) 11/101 (11.9%) ns Miscarriage (Per 5/25 (20%) 4/11 (36%)ns clinical Pregnancy)

Overall clinical pregnancy rates were significantly higher in studypatients (28.1% versus 10.9%; p<0.01). Remarkably, almost one-half ofall pregnancies in the study group were established spontaneously beforeIVF start; however, even within the patients reaching IVF, there was astrong trend towards higher pregnancy rates (20.6% versus 11.9%).

Approximately two months after initiation of treatment the mean DHEA andDHEAS levels at cycle day 2 blood drawing were in the low normal ranges.Few patients reported side effects from DHEA use. These included mildtransient acne on the face, chest or back, oily skin and mild hair loss.No facial or body hair growth was reported, nor was there any deepeningof voice. Some patients reported an increased sense of well-being orincreased libido.

Cox regression of months from initial visit until clinical pregnancy,adjusted for age, race/ethnicity, fertility treatment, and stratifiedfor level of ovarian reserve (POA and DOR), revealed that DHEA treatedpatients had a significantly increased proportional hazards ratio forclinical pregnancy relative to controls (HR 3.8; 95% CI 1.2 to 11.8;p<0.05). FIGS. 6 and 7 show proportional hazard curves of clinicalpregnancy by months from their initial visit. Specifically, FIG. 6 is agraph showing cumulative pregnancy rate of time from initial visit toclinical pregnancy or censor by DHEA for women with premature ovarianaging, and FIG. 7 is a graph showing cumulative pregnancy rate of timefrom initial visit to clinical pregnancy or censor by DHEA for womenwith diminished ovarian reserve. The curves reveal a rapidly separatingincrease in cumulative clinical pregnancies between study and controlgroups from the first month on.

Extended Cox models with correction for time dependent variables “monthsof DHEA use” and “Treatment” did not decrease the proportional hazardsestimation of pregnancy associated with DHEA treatment (HR 4.8; 95% CI1.6 to 14.2; p=0.005).

Discussion of Example 7

A significantly increased pregnancy rate in a group of women with a verypoor prognosis for pregnancy has been determined. A strength of thisstudy is its rather large sample size.

Spontaneous background pregnancy rates in average infertile women occurat an approximate rate of one to two percent per month. Spontaneouspregnancies in women with clear evidence of diminished ovarian functionare obviously an even rarer occurrence. Given the degree of loss ofovarian reserve in this group, a 28.1% cumulative pregnancy rate in apatient population, previously largely referred into oocyte donation, isquite remarkable.

DHEA supplementation can improve ovarian function in women withdiminished ovarian reserve. Study and control patients receivedidentical ovarian stimulation protocols during IVF cycles. IVF protocolsduring the study years 1999-2005 did not significantly change duringthis time. Specifically, the protocols may include administeringmicrodose agonist/gonadotropin stimulations in women with diminishedovarian reserve.

The mechanism of DHEA's action on the ovary remains speculative. DHEAdeclines with age and DHEA supplementation may simply improve thesubstrate pool for steroidogenesis, since DHEA is a precursor hormonefor estradiol and testosterone.

Androgens may, however, influence ovarian follicular growth not only byacting as metabolic precursors for steroid production, but also byserving as ligands for androgen receptors or by other, non-classicalmechanisms. During ovulation induction with exogenous gonadotropins,DHEA is the prehormone for up to 48% of follicular fluid testosterone,which is, in turn, the prehormone for estradiol. There is evidence thatandrogens act, together with FSH, to stimulate folliculardifferentiation. Androgens are also known to promote steroidogenesis andfollicular recruitment and to increase insulin-like growth factor(IGF-1) in the primate ovary. DHEA-treated rat ovaries express elevatedlevels of IGF-1 in pre-antral and early antral follicles.

A transient increase in IGF-1 in patients undergoing exogenousgonadotropin ovulation induction after pretreatment for only eight weeksof DHEA has previously been reported and it was hypothesized that theeffect of DHEA on ovulation induction might have been mediated byincreased IGF-1.

Higher baseline testosterone levels have been associated with improvedIVF outcomes, and higher serum testosterone has been correlated withhigher oocyte numbers retrieved at IVF. Some authors have suggested thatimproved outcomes in women with diminished ovarian reserve afterco-treatment with aromatase inhibitors may be the consequence ofinduction of FSH receptors on granulosa cell by androgens. The resultantovarian response may then lead to improved follicular survival,increased follicle numbers and higher estradiol levels duringstimulation, as classically also observed in polycystic ovarian disease.

Human polycystic ovaries have been described as representing a“stock-piling” of primary follicles, secondary to an alteration at thetransition from primordial to primary follicle. It is possible that DHEAtreatment may create PCO like characteristics in the aging ovary. Longterm exogenous androgen exposure can induce PCO-like histological andsonographic changes. Androgens have been reported to suppress apoptosis.Exogenous DHEA exposure may occur during the first two weeks ofpregnancy.

In summary, a significant increase in the odds of pregnancy among DHEAtreated women has been determined. This increase appears to be rapid inonset and to continue progressively within eight months of initialobservation.

EXAMPLE 8 Decrease Miscarriage Rates

In a further study, women (i.e, women with progressively decliningovarian function) with diminished ovarian reserve were administered DHEAto assess the effect of DHEA on miscarriage rates.

Since women with diminished ovarian reserve produce only few oocytes andembryos, preimplantation genetic diagnosis (PGD) in association with IVFis only rarely indicated, and, indeed, may be detrimental. To accumulatedirect ploidy data on a large enough statistical patient sample is,therefore, difficult. Because spontaneous miscarriage rates arereflective of aneuploidy rates, the study presented herein includespregnancy outcomes after DHEA supplementation from two independent NorthAmerican fertility centers and compares those with age-specific nationaloutcome data after IVF.

Materials and Methods of Example 8

Based on reported clinical experiences, the indications for DHEAsupplementation have changed over the years, with women above age 40since 2007 receiving routine supplementation, and younger womenreceiving supplementation only selectively. This means that under age 40women receive supplementation only if they demonstrate elevatedage-specific baseline follicle stimulating hormone (FSH) levels and havedemonstrated in at least one cycle inappropriately low oocyte yield within vitro fertilization (IVF) following standard ovarian stimulation withgonadotropins.

DHEA supplementation involves the use of pharmaceutical grade micronizedDHEA at a dosage of about 25 mg, three times daily. Patients are on DHEAsupplementation for at least about two months prior to oocyte retrieval.This period of minimal pretreatment is based on the recognition that attwo months pregnancy curves between DHEA pretreated and control patientsstatistically diverge. DHEA is maintained until pregnancy is establishedand is discontinued with positive pregnancy test.

Toronto West Fertility Associates, in Toronto, Canada, started utilizingDHEA independent of the use of DHEA at the Center for Human Reproduction(CHR) in New York, N.Y. In December of 2007, Toronto's medical directorforwarded a detailed electronic record of the center's all-inclusiveDHEA experience for analysis to CHR. This study, therefore, reports onthe miscarriage rate of pregnancies, independently established underDHEA supplementation at both fertility centers, and compares theserates, age-stratified, to miscarriage rates reported in a national IVFdata base in the U.S. for the year 2004. The definitions of clinicalpregnancy, and of miscarriage, used herein follow the reportingrequirements of this national data base, defining a clinical pregnancyas a pregnancy, confirmed by ultrasound examination.

It is important to note that DHEA supplemented patients universallysuffered from severely diminished ovarian reserve. Their pregnancyexpectations were, therefore limited. Patients who conceived a clinicalpregnancy, thus, represented only a minority of DHEA supplementedpatients at both centers.

Miscarriage rates of DHEA supplemented patients were compared withnational IVF outcome statistics, reported annually under Federal mandateby the Centers for Disease Control. The data utilized for this studyreflect 2004 United States national statistics. Pregnancy andmiscarriage rates at the two centers were pooled after confirmation ofhomogeneity of variance. Common odds ratios of the pooled miscarriagerates among age stratified pregnant patients were compared between thepooled rates and the 2004 US national rates utilizing theMantel-Haenszel common odds ratio. Statistical analyses were performedusing SPSS Windows, standard version 15.0.

Results of Example 8

New York reported 40 and Toronto 33 DHEA pregnancies, for a combinedDHEA pregnancy experience of 73 pregnancies. New York reported sixmiscarriages, for a clinical miscarriage rate of 15.0%, and Torontoreported five miscarriages, for a clinical miscarriage rate of 15.2%,for a combined miscarriage rate of 11/73 (15.1%). For analysis, the 2004miscarriage rate in the national U.S. registry of 17.6% was used.

As seen in Table 9 (below) and FIG. 8, miscarriage rates after DHEAsupplementation, stratified for age, were lower at all ages [OR 0.49(0.25-0.94; p=0.04)]. The decrease in miscarriage rate was, however,especially apparent above age 35 years.

TABLE 9 Age-stratified pregnancy and miscarriage rates Age (years) <3535-37 38-40 41-42 >42 DHEA Pregnancies NY 10 5 6 10 9 TO 7 10 13 0 3Miscarriages NY 1 0 0 2 3 TO 1 1 3 0 0 Misc. Rate (%) NY 10.0 0.0 0.020.0 33.3 TO 14.3 10.0 23.1 — 0.0 TOTAL 11.8 6.7 15.8 20.0 25.0 (±95%CI) (15.0) (13.0) (16.0) (25.0) (25.0) NATIONAL Misc. Rate (%) 14.0 17.123.1 36.6 50.1 (±95% CI) (1.0) (1.0) (1.0) (2.0) (5.0) Decrease in Misc.Rate −15.7 −60.8 −31.6 −45.3 −50.1 with DHEA (%) Miscarriage rates afterDHEA supplementation, stratified for age, were lower at all ages [OR0.49 (0.25-0.94; p = 0.04)]. The decrease in miscarriage rate was,however, especially apparent above age 35 years. NY, - Center for HumanReproduction, New York; TO, - Toronto West Fertility Associates,Toronto, CanadaDiscussion of Example 8

The data reported herein demonstrate a significantly diminishedmiscarriage rate in women with diminished ovarian reserve, in comparisonto a standard IVF population, if pretreated for at least two months withDHEA. Specifically, as shown in Table 9, the percentage decrease inmiscarriage rate with DHEA supplementation for women with diminishedovarian reserve under the age of 35 was 15.7, for women between the agesof 35-37 was 60.8, for women between the ages of 38-40 was 31.6, forwomen between 41-42 was 45.3, and for women above the age of 42 was50.1. This effect appears particularly pronounced above age 35 years.

This is a remarkable observation that is further strengthened by thefact that, due to their severely diminished ovarian reserve, the studiedDHEA supplemented women represent a highly unfavorable patientpopulation. It has been reported that women with diminished ovarianreserve experience exceedingly high miscarriage rates, far in excess ofstandard IVF patients with normal ovarian reserve. For example,miscarriage rates of 57.1 percent under age 35 in women with diminishedovarian reserve, 63.5 percent between ages 35 and 40 in women withdiminished ovarian reserve, and as high as 90 percent above age 40 yearsin women with diminished ovarian reserve have been reported. Consideringthe fact that national U.S. IVF data represents only a minority of womenwith diminished ovarian reserve, the finding that DHEA supplementationsignificantly reduced miscarriage rates in all age groups below those ofan average national IVF population is remarkable.

While on first glance the larger degree of reduction in miscarriage ratein older women may surprise, it should not. Aneuploidy rates increasewith age and, indeed, age 35 is generally considered the age cut off,where more aggressive prenatal genetic screening becomes indicated. IfDHEA affects aneuploidy rates, then one would, indeed, expect a muchlarger beneficial effect after, rather than before, age 35, becauseolder women usually produce fewer embryos, and the relative benefit froma decrease in aneuploidy rate on the number of euploid embryostransferred in IVF will, therefore, increase with advancing female age.

Aneuploidy is a frequent finding even in young women. As women age, theprevalence of aneuploidy increases further, at times reaching close to90 percent in women above age 40. Interestingly, women who demonstrateclinical evidence of prematurely aging ovaries do not also demonstrateprematurely enhanced aneuploidy rates. They maintain the expectedage-specific aneuploidy, dictated by their chronological age, andtherefore, experience similar implantation—and pregnancy rates, though,because of decreased oocyte and embryo numbers, reduced cumulativepregnancy rates. It, therefore, should not surprise that women under age35, even though suffering from a significant degree of prematurelydiminished ovarian reserve, did not benefit as much from DHEA as olderwomen.

This study demonstrates a statistical association between DHEAsupplementation and decreased miscarriage rates. The reported dataoffers enough circumstantial evidence to suggest that DHEA bothdecreases miscarriage rates and reduces aneuploidy rates in humanembryos.

The presented data helps to explain why DHEA supplementation increasesegg and embryo quality, improves pregnancy rates and speeds up time toconception. Egg and embryo quality is, of course, at least partially areflection of ploidy. Embryos with less aneuploidy can be expected tolead to more pregnancies, resulting in more, and quicker, conceptions.

The concept of embryo selection by improving ploidy has been the basisfor attempts at improving pregnancy rates and reducing miscarriage ratesvia preimplantation genetic screening (PGS). The utility of PGS hasrecently, however, been seriously questioned since, especially in womenwith only few embryos, the necessary embryo biopsy may cause more harmto pregnancy chances than the potential benefits, derived from embryoselection offer. DHEA supplementation, therefore, may represent a muchsimpler, more cost effective and, most importantly, safer method ofembryo selection for ploidy than PGS.

It should not be overlooked that the here presented study addresses onlyinfertile women with a significant degree of diminished ovarian reserve.As already noted, they represent a very unfavorable patient population,with exceedingly high expected miscarriage rates. However, even thoughinfertile women with normal ovarian reserve have significantly lowermiscarriage rates, they in general still experience higher miscarriagerates than the average population. While the here-reported miscarriagerates in DHEA patients are remarkably low, caution should, therefore, beexercised in concluding automatically that the observed DHEA effect canbe extrapolated to a general population. It is, however, quiteremarkable that the here-reported miscarriage rates in women withseverely diminished ovarian reserve at both study centers, stratified byage, were practically identical to those reported for the generalpopulation.

Based on the hypothesis that congression failure (gross disturbances inchromosome alignment on the meiotic spindle of oocytes) results from thecomplex interplay of signals regulating folliculogenesis (thusincreasing the risk of non-disjunction errors), it has been suggestedthat it may be possible to develop prophylactic treatments that canreduce the risk of age-related aneuploidy. DHEA may, indeed, be a firstsuch drug.

Assuming such a more universal effect of DHEA supplementation onaneuploidy rates, supplementation should also be investigated forinfertile women in general and, maybe, even for normally fertile womenabove age 35, who could receive DHEA as a routine preconceptionsupplement, akin to prenatal vitamins. Should efficacy of DHEAsupplementation in such a general population be proven, the potentialsignificance of such a finding on public health could be considerable.

As stated herein, and supported at least by the examples herein, DHEAsupplementation for at least two months increases egg numbers and eggquality and, therefore, also embryo numbers and quality. DHEA alsoimproves spontaneous pregnancy rates, IVF pregnancy rates, cumulativepregnancy rates and time to conception in prognostically otherwisehighly unfavorable patients. Further, DHEA statistically reducesmiscarriage rates, probably, at least partially, by reducing aneuploidyrates. Moreover, DHEA probably also increases the male/female birthratio. The effects of DHEA increase over time, reaching peaks afterapproximately four to five months of supplementation. It is suggestedthat the peak occurs at four to five months because this time period issimilar to the time period of a complete follicular recruitment cycle.

Example 8 Continued

Background

Dehydroepiandrosterone (DHEA) supplementation may improve selectedaspects of ovarian function in women with diminished ovarian reserve.

DHEA supplementation improves response to ovarian stimulation withgonadotropins by increasing oocyte yield and embryo numbers. DHEAeffects increase over time, reaching peaks after approximately four tofive months of supplementation. DHEA, however, also increases oocyte andembryo quality, spontaneous pregnancy rates in prognostically otherwisehighly unfavorable patients on no further active treatments, pregnancyrates with in vitro fertilization (IVF), time to pregnancy andcumulative pregnancy rates.

DHEA may effect insulin-like growth factors (IGF-1)—mediated. On theother hand, because DHEA effects peak at four to five months, a timeperiod similar to the complete follicular recruitment cycle, we havespeculated that DHEA may effect follicular recruitment, possiblymediated via suppressive effects on apoptosis. Additionally, DHEA mayreduce aneuploidy in embryos.

Since approximately 80 percent of spontaneous pregnancy loss is theconsequence of chromosomal abnormalities, reduced aneuploidy should alsoreduce miscarriage rates. As women get older, and ovarian functionprogressively declines, miscarriage rates rise because of increasinganeuploidy. If DHEA, indeed, were to beneficially affect ploidy, DHEAsupplementation should, as an additional benefit in older women withseverely diminished ovarian reserve, therefore, result in reducedmiscarriage rates.

Since women with diminished ovarian reserve produce only small oocyteand embryo numbers with IVF, preimplantation genetic diagnosis (PGD) inassociation with IVF is only rarely indicated, and, indeed, may bedetrimental. To accumulate direct embryo ploidy data in such patientsis, therefore, difficult. Seeking alternatives, we were attracted by thefact that spontaneous miscarriage rates to such a large degree reflectaneuploidy rates. This study, therefore, presents pregnancy outcomesafter DHEA supplementation from two independent North American fertilitycenters and compares those with age-specific national USA outcome dataafter IVF.

Methods

DHEA supplementation: After approval by the center's institutionalreview board, the Center for Human Reproduction (CHR) in New York Cityhas been utilizing DHEA supplementation in women with diminished ovarianreserve since 2004. Based on reported clinical experiences, theindications for such supplementation have changed over the years: Ininitial stages, only older women, above age 42, were supplemented andonly if they had failed at least one IVF cycle and less than 4 oocyteshad been retrieved in confirmation of ovarian resistance to stimulation.By mid-2005, indications were expanded to all women above age 40 withevidence of ovarian resistance and a history of one failed prior IVFcycle. By early 2006 indications were further expanded to women underage 40 if they demonstrated elevated baseline follicle stimulatinghormone (FSH) levels above 10 mlU/ml and had shown ovarian resistance inat least one failed IVF cycle. By mid-2006 FSH baseline criteria werechanged from absolute FSH elevations to elevations in age-specific FSHlevels. All women above age 40 have been offered routine supplementationsince January 2007, while younger women, under age 40, are continuing tobe only selectively supplemented if demonstrating elevated age-specificbaseline follicle stimulating hormone (FSH) levels and, as previouslyreported, inappropriately low oocyte yield in at least one IVF cycle.

DHEA supplementation in all patients involves oral, pharmaceutical grademicronized medication at a dosage of 25 mg, three times daily (TID).Only morbidly obese women receive an increased daily dosage of 100 mgand no such women were involved in this study. This supplementationdosage was chosen and is continued to be used since DHEA use ahs shownto result in only minor side effects. Limited patient volume and fundingsources have prevented dose response studies and 25 mg DHEA TID dailyhas, therefore, remained the only standard treatment dosage. Patientsreceive at least two months of DHEA supplementation prior to oocyteretrieval, unless they conceive spontaneously during that time period.This minimum pretreatment period is based on the recognition that at twomonths pregnancy curves between DHEA pretreated and control patientsstatistically diverge. DHEA is maintained until pregnancy, and isdiscontinued with second positive pregnancy test.

Collaboration between centers: The utilization of DHEA at the Torontobased center was independently initiated, after that center's medicaldirector (E.R.) at a lecture (by N.G.) learned about the New Yorkcenter's DHEA experience. Toronto's data accumulation was unknown to theNew York center until in December of 2007, unsolicited, a detailedelectronic record of Toronto's DHEA experience was forwarded to New Yorkwith a request for combined analysis. The Canadian data were sequesteredto the New York center's confidential research data base, which isrestricted to one computer. Confidentiality and anonymity of submittedrecords was, therefore, maintained.

Control population: This study reports on miscarriage rates, at bothfertility centers, independently established under DHEA supplementation,and compares these rates, age-stratified, to miscarriage rates reportedin a national USA IVF outcome data base, which involves unselectedinfertility patients. While study populations at the New York andToronto centers, thus, involve women with significantly DOR, thenational control data reflect only a rather small percentage of womenwith this primary diagnosis.

DOR patients have in the past resisted prospective randomization. Tworegistered prospectively randomized, placebo controlled trials, one innew York City and a second in Europe, had to be abandoned for lack ofenrollments (Gleicher N and Barad DH, Unpublished data, 2006 and 2007).In the absence of such prospectively controlled studies, the questionarose how to establish statistically valid controls for observedmiscarriage rates: A control population should involve infertile womenunder treatment. It also should have maximal size, vary in agedistribution (to facilitate age stratification) and be all encompassing(to avoid selection biases). Since here presented DHEA data weregenerated in North America, a USA-based data base, fulfilling all ofthese criteria, was chosen.

The literature does not offer a unified definition of DOR. We define allwomen above age 40 years to suffer from DOR. In women under age 40 thediagnosis is only reached if age-specific ovarian function parameters.

Definitions of clinical pregnancy and of miscarriage follow thereporting requirements of this national data base, defining clinicalpregnancy, as confirmed by ultrasound.

Since patients at both study centers, as a prerequisite to DHEAsupplementation, had to suffer from DOR, their expectation of pregnancysuccess is very limited. Even considering a higher conception rate insuch patients after supplementation with DHEA, conceptions will occur inonly a small minority of DHEA supplemented cycles. The here reportednumber of consecutive pregnancies, therefore, represents a range ofapproximately 450 to 570 initiated DHEA treatment cycles.

Statistics: Miscarriage rates of DHEA supplemented patients werestatistically compared with national IVF outcomes, reported annuallyunder federal mandate by the Centers for Disease Control and Prevention,U.S. Department of Health and Human Services. The data utilized ascontrols for this study reflect 2004 United States IVF statistics,report cycle numbers, pregnancy percentages and live birth percentages,stratified for age. These detailed national data allowed calculation ofnumber of clinical pregnancies and number of live births for each agegroup, since neither is offered in the original data set. We thensubtracted live births from pregnancies, to derive number of failedpregnancies (i.e., all failed pregnancies were for purpose of this studyconsidered miscarriages) overall, and in each age category. Counts ofpregnancies and miscarriages were then entered into a series of two bytwo tables, stratified by age, and using the cross tabulation module ofSPSS 15.00.

Pregnancy and miscarriage rates at both fertility centers were pooledafter confirmation of homogeneity of variance. Common odds ratios of thepooled miscarriage rates among age stratified pregnant patients werecompared between the pooled centers and 2004 national rates, utilizingthe Mantel-Hänszel common odds ratio (tests for homogeneity of the oddsratio across layers were not significant, meeting assumption for use ofthis test) and using dichotomous exposure (DHEA versus controls) anddichotomous outcomes (live births versus spontaneous miscarriages),stratified by five age categories.

A secondary statistical analysis of the data was performed, byrecalculating for all five investigated age groups (<35, 35-37, 38-40,40-42 and >42 years) expected miscarriage rates for both patient groups,equalized for size. Both statistical analyses are presented in sequenceand were performed using SPSS Windows, standard version 15.0.

Institutional Review Board: The investigation of DHEA in women with DORhas been repeatedly approved by the center's Institutional Review Board(IRB). Since the here reported study only involved the evaluation of(electronic) medical records, and maintained their confidentiality, thehere presented study, based on a patient consent signed at time ofinitial registration, did not require further IRB approval. Aconfirmatory written statement from the chairman of the IRB is availableupon request.

Results and Discussion

New York reported 40 and Toronto 33 DHEA pregnancies, for a combinedDHEA pregnancy experience of 73 pregnancies. Among those pregnancies,New York registered six and Toronto five miscarriages, for clinicalmiscarriage rates of 15.0% and 15.2%, respectively, and a combinedmiscarriage rate of 11/73 (15.1%). In comparison, the total 2004miscarriage rate in the national USA registry was 17.6%. The odds ratioand 95% confidence interval (CI), stratified for age, that a woman wouldmiscarry was, thus, statistically significantly lower after DHEAsupplementation [OR 0.49 (0.25-0.94; p=0.04), suggesting a reduction inmiscarriage risk of approximately 50 percent (data not shown;Mantel-Hänszel, distributed as Chi-square with one degree of freedom,4.285; p=0.038).

When expected miscarriage rates were compared in both patient groups,equalized for number of patients, women after DHEA supplementationdemonstrated even more significant reductions in miscarriage rate(p<0.0001) suggesting an almost 80% reduction in miscarriage risk (datanot shown; Mantel-Haenszel, distributed as Chi-square with one degree offreedom, 12.482; p<0.0001).

Differences between DHEA treated patients and the national IVF databecame even more obvious after age-stratification. Table 9 and FIG. 8summarize age-specific rates in numerical and graphic formats:Miscarriage rates at all ages were lower in DHEA patients than in the2004 national IVF data. Those differences were, however, only after age35 years pronounced.

Here reported data, after DHEA supplementation, demonstrate in womenwith DOR significantly lower miscarriage rates than in a standard IVFcontrol population, a finding particularly pronounced above age 35years. This remarkable observation is further enhanced by the wellrecognized and reported excessive miscarriage risk of women with DOR.Levi et al, for example, reported that women with DOR experiencemiscarriage rates far in excess of standard IVF patients with normalovarian reserve: 57.1 percent under age 35; of 63.5 percent between ages35 and 40 and as high as 90 percent above age 40 years.

Patients in the here reported study that suffered from DOR is bestdocumented by them receiving DHEA supplementation. Under our center'sDHEA protocols, except for women above age 40 years, DHEAsupplementation is offered only to women who have failed at least oneprior IVF cycle with retrieval of less than four oocytes and, therefore,have been designated resistant to ovarian stimulation. Moreover, youngerwomen receive DHEA supplementation only if they also demonstrateelevated age-specific FSH levels. Finally, DHEA supplementation isvoluntary, allowing for the assumption that more severely compromisedpatients, with poorer past IVF experiences, will more likely choosesupplementation.

In contrast, USA IVF outcome data only in a minority represent womenwith diminished ovarian reserve. As Levi et al demonstrated, controlpopulations, therefore, should demonstrate significantly lowermiscarriage rates than our study patients. The finding that women onDHEA supplementation demonstrate in all age groups, but especially aboveage 35, significantly lower miscarriages than the much more favorablenational IVF population is, therefore, noteworthy.

That this difference is less obvious under age 35, only strengthens thevalidity of the here utilized controls. Indeed, the larger degree ofreduction in miscarriage rates in older women should not surprise:Aneuploidy rates increase with age, and age 35 is generally consideredthe cut off, when invasive prenatal genetic prenatal screening becomesindicated. Assuming a beneficial effect of DHEA on aneuploidy rates, alarger effect after age 35 should, therefore, be expected.

Levi et al, reported in women with diminished ovarian reserve above age40 an approximately 90% miscarriage rate. Since older women producefewer embryos, the relative benefits from decreases in aneuploidy rateon number of euploid embryos, transferred into the uterus, will increasewith advancing female age.

Aneuploidy is, however, even in young women a frequent finding. In womenwith diminished ovarian reserve Levi et al reported an almost 60 percentmiscarriage rate under age 35 years. As women physiologically age, theprevalence of aneuploidy continues to increases, reaching close to 90percent in the mid-40ies. Premature ovarian aging, however, does notprematurely enhance aneuploidy rates, and instead maintains expectedage-specific aneuploidy rates. Though demonstrating features of ovarianaging, affected women, therefore, still experience age-appropriateimplantation—and pregnancy rates. Because of decreased oocyte and embryoyields, they, however, do demonstrate reduced cumulative pregnancyrates. Even though significantly affected by prematurely diminishedovarian reserve, a smaller benefit from DHEA under age 35 in our studypopulation should, therefore, not surprise.

By demonstrating in a very high risk population for spontaneouspregnancy loss a statistical association between DHEA supplementationand decreased miscarriage rates, this study does not proof causation.The study, therefore, does not prove that DHEA decreases miscarriage oraneupoidy rates in human embryos. The here reported data, however, offerenough circumstantial evidence to suggest that DHEA may, indeed, exertboth of these effects and, therefore, warrant further investigations. Asuggestion of improved euploidy after DHEA supplementation was, afterall, also observed in human embryos.

Our center's miscarriage rates in women with DOR, prior to introductionof DHEA supplementation, were higher than the national rate seen in thehere utilized control population. The program's pregnancy rates in thesewomen were then only in low single digits. The gradual introduction ofDHEA supplementation between 2004 and 2007 progressively improvedpregnancy rates at our center. Increasing pregnancy numbers anecdotallysuggested a concomitant decline in miscarriage rates. This observation,in turn, lead to the previously noted investigation of aneuploidy ratesin embryos after DHEA supplementation, which, though statisticallyunderpowered, was supportive of a beneficial DHEA effect on ploidy.

The New York center's pregnancy and miscarriage data, alone, were,however, not large enough to allow for statistically valid conclusionsabout factual miscarriage rates. Such conclusions became possible, oncethe independently collected Toronto data became available, andstatistical analysis demonstrated that the two data sets could beunified. At this point the question arose how to control the twocenters' miscarriage experiences. A statistical comparison to a largeand unselected, national data set appeared appropriate.

While such a comparison cannot replace the gold standard of studydesign,—the prospectively randomized and placebo controlled study, thehere presented data, nevertheless, offer valuable new information. We inthis study used carefully vetted statistical methodologies, which areappropriate for the kind of comparisons offered. Moreover, we evenperformed a second statistical analysis, based on a differentstatistical model, which suggested an even bigger beneficial statisticaleffect of DHEA supplementation, increasing the potential benefit from anapproximately 50 percent to an approximately 80 percent reduction inmiscarriage risk.

Whether the benefit of DHEA supplementation is, indeed, 50 or 80 percentcan as of this moment not be ascertained with certainty, but also shouldnot matter. What seems of importance is the observation that DHEAsupplementation, at least in women with DOR, who characteristicallydemonstrate abnormally high miscarriage rates, appears to significantlyreduce the risk for spontaneous pregnancy loss.

Our here presented data may help to explain why DHEA supplementationincreases egg and embryo quality, improves pregnancy rates and speeds uptime to conception. Egg and embryo quality is, of course, at leastpartially a reflection of ploidy. More euploid embryos will lead to morepregnancies, thus shortening time to conception.

It is important to note that DHEA supplementation, as described, appearssafe and results in only minor side effects. Since DHEA is a mildandrogen but is converted into testosterone (and estradiol), it shouldnot surprise that observed mild side effects, such as oily skin, mildacne vulgaris and hair loss are mostly androgenic in nature.

Embryo selection and improving embryo ploidy have been the rational forattempts at improving pregnancy rates and reducing miscarriage rates viapreimplantation genetic screening (PGS), a concept recently seriouslyquestioned. Here presented data suggest that DHEA supplementation mayresult in more cost effective improvements in ploidy without laboratoryintervention.

Though infertile women with normal ovarian reserve experiencesignificantly lower miscarriage rates than DOR patients, they stillexperience higher miscarriage rates than average populations. Herereported miscarriage rates in DHEA treated DOR patients are, therefore,remarkably low and practically identical to those reported for generalpopulations. Caution should, however, nevertheless, be exercised inconcluding that observed DHEA effect can automatically be extrapolatedto normal, fertile populations, though such a possibility deservesfurther investigation. If confirmed, one could perceive DHEA as aroutine preconception supplement, akin to prenatal vitamins, even inwomen with no fertility problems.

Conclusions

Based on the hypothesis that major disturbances in chromosome alignmenton the meiotic spindle of oocytes (i.e., congression failure) resultfrom complex interplay of signals, regulating folliculogenesis(increasing the risk of non-disjunction errors), Hodges et al suggestedthat it may be possible to develop prophylactic treatments which canreduce the risk of age-related aneuploidy. This study suggests that DHEAmay, indeed, be a first such drug.

Should efficacy of DHEA supplementation be proven not only in infertilepatients but also in general populations, the potential significance onpublic health could be considerably and by far exceed the more imminentutilization of DHEA in fertility practice.

EXAMPLE 9

Amidst considerable gains in the treatment of infertility, diminishedovarian reserve (DOR), whether due to physiologic aging of the ovariesor premature ovarian aging (POA), represents one of the few unresolvedproblem of modern infertility care. Indeed, as treatment success withother infertility problems has improved, POA patients increasinglyappear to concentrate in infertility centers, and women above age 40years have become the proportionally most rapidly growing age group inU.S. maternity wards, concomitantly graying the population underinfertility treatments.

Dehydroepiandrosterone (DHEA) supplementation of women with DOR maypositively impact ovarian function by increasing oocyte yields afterstimulation with gonadotropins. We confirmed, and expanded on thisobservation by demonstrating that DHEA also improves egg and embryoquality, pregnancy rates, time to conception and reduces miscarriagerates.

Women with significant degrees of DOR usually have limited time left toconceive with use of autologous oocytes and, as two recently cancelledclinical trials (in the U.S. and Europe) demonstrate, are, therefore,reluctant to enter prospectively randomized studies that may assign themto placebo. All so far published DHEA data are, therefore, either cohortor case controlled studies, representing lower levels of evidence.

In the absence of prospectively randomized, placebo controlled studies,we searched for alternatives. Since DHEA apparently increases oocytesyield, it, likely, positively affects ovarian reserve (OR). OR hastraditionally been investigated utilizing baseline follicle stimulatinghormone (FSH). More recently, anti-Müllerian hormone (AMH) has, however,been suggested as a more specific reflection of OR. Its utilization inassociation with prematurely DOR has been advocated. This study,therefore, utilized AMH to assess OR following DHEA supplementation.

Materials and Methods Of Example 9

The study is a cross-sectional analysis of 120 consecutive women withDOR in whom AMH levels were evaluated as a reflection of OR. Theypresented during 2007/8 to our center for initial infertilityconsultation. First AMH levels obtained were used for INITIAL analysis.Post DHEA initiation, exposure to the supplement ranged from 34 to 119days (mean 73±27 days). Women with two or more consecutive AMH levelscomprised patients in the longitudinal study evaluation of OR afterinitiation of DHEA supplementation.

Our center defines DOR in women under age 40 by elevated age-specificFSH levels, as previously reported in detail, or by universal AMH levelsbelow 0.8 ng/ml, which approximately correlate to an FSH of 11.0 mIU/ml.Since OR declines with advancing female age, women above age 40 areuniformly assumed to suffer from DOR. Age-specific FSH levels have inassociation with in vitro fertilization (IVF) been demonstrated todiscriminate between oocytes yields.

FSH and estradiol were evaluated by standard enzyme-linkedimmunoabsorbent assay (ELISA; AIA-600II, Tohso, Tokyo, Japan). Onlyresults in assay range were considered for statistical evaluation. AMHlevels were also obtained by ELISA. In short, the DSL-14400 activeMüllerian Inhibitig Substance/Anti-Müllerian Hormone (MIS/AMH)Enzyme-Linked immunoabsorbent (ELISA) was utilized (Diagnostic SystemsLaboratories, Inc. Webster, Tex. 77598-41217, USA). This is anenzymatically amplified two-site immunoassay, which does not cross reactwith other members of the TGF-β superfamily, including TGF-β1, BMP4 andACT. Theoretical sensitivity, or minimum detection limit, as calculatedby interpolation of the mean plus two standard deviations (SD) of eightreplicates of the 0 ng/ml MIS/AMH Standard, is 0.006 ng/ml. Intra-assaycoefficient of variation for an overall average AMH concentration is ≦20percent.

Since 2007, DOR patients are at our center, before being advanced intoIVF, for at least two months supplemented with pharmaceutical grade,micronized and pharmacy compounded DHEA at a dosage of 25 mg TID. DHEAis continued throughout all IVF cycles until conception (second,normally rising positive pregnancy test) or until patients discontinuetreatment with autologous oocytes.

The study population was age-stratified under and above age 38 years,and further stratified, based on whether clinical pregnancy had beenachieved or not. Age 38 was chosen as cut off because it has beenreported to represents the beginning of accelerated decline in ovarianreserve.

Data are shown as means±standard deviation (SD) or as raw numbers andpercentages. Data analysis was performed using SPSS windows, version17.0. Demographic and biochemical data were analyzed with paired orunpaired Student's t-test. A generalized linear model was performed toevaluate the interaction of pregnancy status with days of DHEA exposure,adjusted for age at start of treatment.

DHEA utilization at our center was initially approved by the center'sinstitutional review board (IRB) under various study protocols. Afterpublication of a number of studies, the utilization of DHEA was in 2007routinely expanded to all women above age 40 and to younger women withevidence of diminished ovarian reserve. Patients, nevertheless, arestill mandated to sign a DHEA-specific informed consent, which, amongstother facts, advises them that DHEA by prescription is not approved bythe Food and Drug Administration to treat DOR, and is in the UnitedStates commercially available as a food supplement without prescription.

The center's IRB allows for expedited review of studies, which onlyinvolve review of medical records since all patients at initialconsultation sign an informed consent, which allows for such reviews forresearch purposes as long as the medical record remains confidential andthe identity of the patient is protected.

Results of Example 9

The patient population comprised 74% Caucasians, 11% African Americanand 15% Asian patients. A large majority (85%) were recorded with aprimary diagnosis of DOR, 3% with male factor infertility and 12% withtubal factor.

Table 10 below summarizes the characteristics of the study populations,separately for cross-sectional (n=120) and longitudinal assessments(n=55). Obviously, low baseline AMH and high FSH levels are confirmatoryof significant DOR in the study population. Age ranges also confirm thatthe younger patient population, indeed, does reflect relatively younginfertility patients and, therefore, with a considerable prevalence ofPOA, while the older age group in principle represents women above age40 years.

TABLE 10 Characteristics of study patients Cross-sectional LongitudinalStudy Group Study Group All <38 years ≧38 years p-value Number ofpatients 120 55 18 37 Age (years); mean ± SD  39 ± 3.9  39 ± 3.1 34.9 ±3.1  42.1 ± 1.2  <0.001 AMH (ng/ml); Mean ± SD 0.32 ± 0.20 0.22 ± 0.220.20 ± 0.16 0.23 ± 0.17 n.s. Baseline FSH (mIU/ml); Mean ± SD 15.9 ±14.1 15.4 ± 9.1  14.2 ± 8.2  18.0 ± 10.8 n.s. Estradiol (pg/ml) Mean ±SD 60.0 ± 50.0 52.3 ± 28.6 56.1 ± 13.6 53.2 ± 36.6 n.s. Maximal DHEA-S(microg/dL)* Mean ± SD 474 ± 145 476 ± 180 475 ± 224 478 ± 180 n.s.*First value obtained 30 days after initiation of DHEA supplementation

Cross-sectional evaluation of the whole patient population (FIG. 9)demonstrates, unadjusted for age, AMH levels as a function of length ofDHEA supplementation. FIG. 9 very clearly demonstrates a steady increasein AMH over time until 120 days after initiation of DHEA (p=0.002). Age(p=0.007) and length of DHEA supplementation (p=0.019) wereindependently associated with AMH levels. Younger women, under age 38years, demonstrated higher AMH levels from baseline, and proportionallyimproved AMH levels over time after initiation of DHEA more than olderwomen at, or above, 38 years.

Very similar results were obtained in longitudinal evaluation: Here, AMHlevels improved from 0.22±0.22 ng/ml at baseline, before start of DHEA,to 0.35±0.03 ng/ml at highest measured peak, an almost 60 percentimprovement in mean (p=0.0001).

Amongst 55 women who had undergone IVF, by time of analysis, 13 (23.64%)conceived a clinical pregnancy. FIG. 10 demonstrates a comparison of AMHlevels after DHEA supplementation in women who did and did not conceive.As the figure demonstrates, those who conceived demonstrated asignificantly better AMH response, following DHEA supplementation, thanunsuccessful patients, whose AMH response remained flat (interaction ofpregnant versus non-pregnant, Wald Chi Square 11.6; df=1; p=0.001).

Discussion of Example 9

By assessing changes in AMH levels, this study for the first timepresents objective evidence that DHEA supplementation positively affectsdiminished ovarian reserve. In concordance with our prior clinicalobservations, this DHEA effect is visible in younger and older ovaries(FIG. 9), though is more pronounced in younger women with POA.

This study also strongly suggests that observed improvements in OR afterDHEA supplementation lead to better pregnancy rates. As Table 10demonstrates, AMH and FSH levels in the here-utilized study populationare highly confirmatory of a significant degree of DOR.

Indeed, over half of the here-investigated patients consulted with ourcenter for the first time after receiving advice elsewhere todiscontinue fertility attempts with autologous oocytes and proceed intoegg donation.

That in such an adversely selected patient population approximately onein four women still conceived with use of autologous oocytes is, initself, a remarkable accomplishment. It is, however, especiallyremarkable that, as FIG. 10 demonstrates, the ovarian response patternto DHEA is so dramatically different between those women who ended upconceiving and those who did not. While those with future pregnanciesdemonstrated remarkable improvements in AMH levels, unsuccessful womendemonstrated generally no response whatsoever to DHEA. They, thus, forpractical purposes can be seen as a control group: where DHEA does notimprove OR, as indicated by generally flat AMH levels, pregnancy is veryunlikely.

While associations in non-randomized studies always have to be viewedwith caution, here reported results appear convincing. First, theobserved pregnancy rate corresponds well to previously publishedclinical observations at our center, following DHEA supplementation.Improved pregnancy rates following DHEA have since also been reported byinvestigators in Greece and Canada.

While we and others have in the past speculated about possiblemechanisms, why DHEA would improve conception rates in women with DORhas remained unknown. We recently suggested that at least part of DHEA'seffect may be a reduction in oocytes and embryo aneuploidy. This study,however, for the first time offers a more direct and clinicallypractical explanation for DHEA effects in women with DOR.

The concept of OR has been based on a presumed remaining follicular poolwithin ovaries. As this pool shrinks, OR, and with it female fecundity,decline. In the process, the size of immature follicular cohorts,recruited each month, also declines. As cohorts decline in size, smallerand smaller follicle numbers reach gonadotropin sensitivity—the laststage of follicular maturation. As a consequence, follicle numbers andoocytes yield in IVF decline with advancing female age, as does femalefecundity in general.

In fertility practice follicle numbers and oocytes yield are consideredultimate measures of OR. Indeed, AMH is increasingly considered a betterreflection of OR because it better predicts oocytes yield in IVF thanFSH. AMH, a dimeric glycoprotein and a member of the transforming growthfactor (TGF) superfamily, is exclusively produced by granulose cells ofearly developing follicles, from primary to antral follicle stages. AMHis, thus, reflective of small, pre-antral follicles but not of the laterstage follicular pool, better represented by FSH levels. AMH appears tobetter reflect total quantity and, possibly, quality of the remainingfollicular pool, and, therefore, to be a better marker of decliningreproductive age, an observation which potentially explains how DHEAaffects ovarian function.

By demonstrating improving AMH levels, this study suggests that inselected patients with DOR, DHEA progressively improves OR at follicularstages at which AMH is produced. This means that over time DHEAincreases the pool of follicles up to pre-antral stage, in this studycausing a steady improvement in AMH up to 120 days post DHEA initiation.In prior clinical studies, with longer follow up periods, wedemonstrated that follicular numbers and oocytes yield increase up toapproximately five months of DHEA supplementation, equal to theapproximate time period from primordial stages to gonadotropinsensitivity.

Combined, these observations suggest two possible mechanisms by whichDHEA exerts its effects, both reflective of impacts on the follicularmaturation cycle and improvements in number of AMH producing follicles:DHEA either positively affects recruitment from the dormant follicularpool or it progressively reduces apoptosis of originally recruitedfollicles, which represents the primary process by which originallyrecruited follicles are eliminated during follicular maturation. Eitherway, progressively more pre-antral follicles accumulate, resulting inthe here documented increase in AMH over time from initiation of DHEAsupplementation.

The effect of DHEA on follicular recruitment has not been investigated.Androgens, in general, appear, however, capable of positively affectingfollicular recruitment in the mouse.

Similarly, nothing is known about DHEA effects on apoptosis, andandrogens, in general, have been reported to have both enhancing andsuppressing effects on ovarian granulose cell apoptosis.

Our previously published clinical observations suggested thatapproximately two months of DHEA supplementation were required beforestatistically significant differences in outcomes could be observed.FIG. 10 suggests that beneficial effects of DHEA may already becomeapparent even earlier, and may be reflected in spontaneous pregnancieswe and others have reported in a small number of prognostically highlyunfavorable patients, preceding other therapeutic interventions.

While improving AMH levels in women with DOR appear closely associatedwith pregnancy success, AMH is, unfortunately, not sensitive enough topredict who will or will not conceive.

Pregnancies can even be established at undetectable AMH levels. Thismeans that AMH levels alone will not allow discrimination between whodoes and does not deserve further infertility treatments.

As this study, however, demonstrates, AMH offers objective evidence forthe therapeutic efficacy of DHEA in women with DOR, and especially underage 38 years. Moreover, a good AMH response to DHEA supplementationclearly discriminates between good and poor prognosis patients inregards to pregnancy success. This information alone will greatlyimprove patient counseling in women with significant DOR. We arecurrently investigating other markers of OR in attempts to even betterpredict success of DHEA supplementation and, thus, avoid such treatmentin women who will not improve pregnancy chances in response to DHEAsupplementation.

EXAMPLE 10 AMH Levels

Introduction of Example 10

Functional ovarian reserve (OR) declines with advancing female age(Knauff et al. 2009); yet, ovarian function tests traditionally utilizecut-off values for normal ovarian function in age-independent ways. Forexample, with the most frequently utilized OR-test, follicle stimulatinghormone (FSH), cut-off values of 10.0-12.0 mIU/mL have traditionallybeen considered the upper limit of normal (Barad et al. 2007).

More recently, anti-Müllerian hormone (AMH) has found increasingapplication in determining OR (Ebner et al. 2006; Fleming et al. 2006;Nelson et al. 2007; Broer et al. 2009; Carlsen et al. 2009; Knauff etal. 2009; Nelson et al. 2009). We demonstrated that, while AMH and FSHcorrelate (Singer et al. 2009), AMH is superior to FSH in predictingoocytes yields (i.e. OR) and IVF outcomes (Barad et al. 2009). Gnoth etal. suggested that a minimum level of 1.26 ng/mL denotes diminishedovarian reserve (DOR) in women of all ages (Gnoth et al. 2008).

We previously pointed out that, in determining OR, age-specific (as-)FSH levels are preferable to non-age-specific (nas-) cut-off values,discriminate between better and poorer oocyte yields in association within vitro fertilization (IVF), and allow for a more accurate diagnosis ofDOR, especially in younger women under age 38 years (Barad et al. 2007).In similar fashion, one can expect as-AMH levels to be superior tonas-cut-off levels. Such as-cut-offs have, however, so far not beendefined.

This study, therefore, analyzed as-cut-offs in an infertility populationof women and attempted to determine to what degree as-AMH levels coulddiscriminate between women with better and poorer OR, based on oocytesyields in IVF.

Materials and Methods Of Example 10

778 consecutive female patients in 2007 and 2008 represented anunselected initial study population. To define as-AMH levels based on95% CI cut off values, women with obviously elevated baseline FSH above12.0 mIU/ml were eliminated from establishing as-AMH cut off values,leaving 206 patients for statistical analysis. They were separated intofour age categories: below 30 years, 31 to 35 years, 36 to 40 and 41years and above.

Within each age group, as-AMH levels were determined, based on the 95%confidence intervals (CI) of the mean, using first AMH sampling resultsat the center.

Since 2007, our center assesses AMH routinely at time of a new patient'sinitial blood draw. A total of 288 amongst the original 778 women hadreached IVF by time of data analysis. They were utilized to analyzeoocytes yields in reference to the as-OR parameters AMH and FSH.

Patients sign at time of initial consultation an informed consent, whichpermits for use of data from their medical record for clinical researchpurposes, as long as the medical record remains confidential, and theidentity of the patient remains protected. Such record-based studies arethen only subject to an expedited review process by the InstitutionalReview Board.

Race/ethnicity of patients are determined at initial consultation.Clinical circumstances and infertility diagnoses are periodicallyreevaluated as new clinical and laboratory data are obtained. Selectedclinical patient data are, aside from each patient's medical record,also maintained in the center's electronic research data base, which iswritten in Microsoft Access, and is only accessible to authorizedclinical investigators.

Normal as-AMH levels were defined as within the 95% CI of each agegroup. DOR was diagnosed if AMH levels were under the lower cut off foran age group's normal range. A possible diagnosis of polycystic ovariansyndrome (PCOS), and, therefore, of potential OHSS risk, was consideredat as-AMH levels above the upper 95% CI for an age group.

The 288 women who by time of data analysis had reached a first in vitrofertilization IVF) cycle, were separately analyzed from the whole studygroup Like the complete study population, they were divided into thesame age categories. Oocyte yields were then assessed within each agecategory, based on whether a patient demonstrated normal, low or highas-AMH.

As previously reported (Barad et al. 2009), a commercially availableenzyme-linked immunoabsorbent assay (ELISA) is utilized to assess AMH.In brief, this is the DSL-10-14400 active Müllerian InhibitingSubstance/Anti-Müllerian Hormone (MIS/AMH) enzyme-linked immunoabsorbent(ELISA) (Diagnostic Systems Laboratories, Inc. Webster, Tex. 77598-4217,USA), an enzymatically amplified two-site immunoassay, which does notcross-react with other members of the TGF-β superfamily, includingTGF-β1, BMP4 and ACT (Kevenaar et al. 2006). Theoretical sensitivity, orminimum detection limit, calculated by interpolation of mean plus twostandard deviations of eight replicates of the 0 ng/mL MIS/AMH Standard,was 0.006 ng/mL. Intra-assay coefficient of variation for an overallaverage AMH concentration was ≦10 percent (Kevenaar et al. 2006).Results are presented in ng/mL, with a conversion factor×7.14 to pmol/L(Ebner et al. 2006).

In addition to AMH, OR was assessed via cycle day 2/3 FSH and estradiollevels, obtained in the cycle preceding IVF. Both hormones were assessedutilizing an automated chemiluminescence system (ACS: 180, Bayer HealthCare, Tarrytown, N.Y.)

Initial ovarian stimulation protocols of patients are principallydetermined by their age, with secondary modifications made based onovarian function assessments. With presumed normal ovarian reserve,women up to age 38 years are routinely stimulated in a long agonistprotocol with 150 to 300 IU of human menopausal gonadotropin (hMG)daily. Above age 38, or if women are considered to suffer from DOR ateven younger ages, routine stimulation calls for a microdose agonistprotocol with at least 450 IU of gonadotropins daily, most given asfollicle stimulating hormone (FSH), but 150 IU given as hMG, aspreviously reported (Karande et al. 1999).

IVF cycles are conducted in routine fashion. In brief, human chorionicgonadotropin (hCG) is administered after leading follicles exceedaverage diameters of 18 mm, and oocyte retrievals under ultrasoundcontrol take place approximately 34 hours after hCG. Retrievedfollicular fluids are immediately transferred to the embryologylaboratory, where oocyte yields are determined.

All data are expressed as mean±standard deviation (SD). Variables thatdid not conform to normality were log converted and back-transformed.They are presented as means and 95% CI of the mean. A p-value<0.05 wasconsidered statistically significant.

Differences between normally distributed variables were tested withanalysis of variance or co-variance. Differences between groups ofvariables, not conforming to normality, were tested with theMann-Whitney test and p<0.05 was, here too, considered statisticallysignificant. All analyses were carried out utilizing SPSS software forWindows, version 17.0, 2005 (SPSS Inc. Chicago, Ill.)

Results of Example 10

FIG. 11 is a table showing patient characteristics. IVF patients did notdiffer statistically in any parameter from the whole study group. CI isconfidence interval; DOR is diminished ovarian reserve; POA is prematureovarian aging.

FIG. 11 summarizes patient characteristics separately for the total,initially presenting patient population of 778 women, and for 288patients who reached IVF. Amongst the total population, 67.1% wereCaucasian, 13.9% African and 19.1% Asian and the racial distributionamongst IVF patients was almost identical.

Primary infertility diagnoses were also very similar in both patientgroups. In the total population this included the following: DOR and/orpremature ovarian aging (POA, 51.4%), tubal infertility (20.2%), malefactor (13.9%) and “other” (6.6%). IVF patients demonstrated an almostidentical distribution (FIG. 11).

FIG. 14A is a graph showing as-AMH levels (Anti Mullerian Hormoneng/ml). The figure demonstrates means and 95% CI for AMH (upper panel)relating to female age. FIG. 14B is a graph showing as-FSH levels(Follicle Stimulating Hormone mlU/ml). The figure demonstrates means and95% CI FSH (lower panel) relating to female age. In accordance withbetter specificity of as-AMH than as-FSH, reported here (see discussion)and elsewhere (Barad et al., 2009), as-ranges for AMH are narrower thanthose of as-FSH.

FIG. 14 demonstrates that both, AMH (upper panel) and FSH (lower panel),statistically to a significant degree change with age (p<0.001, each).The figure, however, also demonstrates that as-hormone ranges arenarrower with AMH than FSH. Moreover, with both hormones the most narrowrange and, therefore, highest specificity is reached at approximatelyage 35 years, with as-ranges from that point on expanding with youngeras well as older ages.

FIG. 12 is a table showing hormone levels among 206 patients with normalbaseline FSH. AMH levels decrease significantly between age categories(p<0.001), while FSH increases (p=p<0.001) and estradiol remainsunchanged. FIG. 12 summarizes lower and upper cut off values at variousages.

FIG. 15 is a table showing the definition of as-AMH (Anti MullerianHormone). The figure demonstrated means and 95% CI of AMH for 4 agegroups. AMH levels declined significantly with age (p<0.001).

When age categories are set for AMH (FIG. 15), statistically robustcut-off values, representing the 95% CI for any given age group, can beestablished. FIG. 12 offers details: Means of as-AMH, as well as upperand lower cut offs, decrease significantly from age-bin to age-bin aswomen age. Mean levels decline from 3.8 ng/mL below age 30, to 2.0 ng/mLat age 30 to 34, to 0.9 ng/mL at 35 to 40 and to 0.4 ng/mL at age 40years.

As FIG. 12 demonstrates, normal as-AMH ranges are under age 30 3.1 to4.6 ng/mL, at ages 31 to 35 years 1.5 to 2.7 ng/mL, between ages 36 and40 above 0.8 to 1.1 ng/mL and from age 41 on 0.2 to 1.0 ng/mL.

Based on these criteria, 138/287 (48.1%) women, who reached IVF (onepatient had no recorded pre-IVF AMH value), demonstrated abnormally low,57 (19.9%) normal and 92 (32.1%) abnormally high, as-AMH levels.

FIG. 16A is a graph showing oocyte yields at different ages and AMHlevels. Upper panel [A] demonstrates oocytes yields with normal and high(red) and abnormally low (blue) AMH. The figure suggests that oocyteyield among women with normal and high as-AMH decline only mildly up toage 30, when their decline accelerates. In contrast women withabnormally low as-AMH appear to decline steadily till age 39, when thedecline appears to flatten. Differences were, however, not statisticallysignificant. FIG. 16B is a graph showing oocyte yields at different agesand AMH levels The lower panel [B] defines oocytes yields in eachage-group based on abnormally low (blue), normal (red) and abnormallyhigh (beige) as-AMH levels. Normal was defined as the 95% CI for a givenage-group. The patient group above age 40 years is too small to offerstatistically valid conclusions. Table 13 offers further statisticaldetails.

FIG. 13 is a table showing oocyte yields among patients reaching IVF.Superscripts denote significant difference (p<0.05) from designatedcolumn within age categories: ^(a) denotes low as-AMH; ^(b) normalas-AMH; ^(c) high as-AMH; Total oocytes yields in women with as-normaland high AMH were significantly higher than those in women as-low AMH(F=78.9, df 1; p<0.00001).

FIG. 16 and FIG. 13 summarize oocytes yields in the four age categories.Oocyte numbers declined overall with advancing female age (p<0.001).

FIG. 16, however, defines the subtleties of this decline afteradjustment for age: FIG. 16A (upper panel) demonstrates oocytes yieldsin the four age categories, depending on low (in blue) or combinednormal and abnormally high (in red) as-AMH. As the figure demonstrates,oocyte yields with abnormally low as-AMH were in all age-categories tillage 40 significantly lower than with normal (and high) AMH (p<0.001; forfurther statistical detail, see FIG. 13). Indeed, oocytes yields,overall, were 5.4-times (95% CI 4.1-6.8) higher in women with normal andabnormally high as-AMH in comparison to those with subnormal levels.

FIG. 16B further defines these data because the figure separates oocytesyield in each age category by abnormally low (blue), normal (red) andabnormally high (beige) as-AMH: At youngest ages (<30 years) as-AMHstatistically differentiates low yields with abnormally low AMH fromvery similar higher yields with normal and even abnormally high as-AMH.In this age group excessively high AMH, however, does not define a highrisk group for excessively high oocytes yields since yields do notdiffer between normal and high as-AMH. FIG. 13 summarizes thestatistical details.

This picture, however, changes in the next two age-categories, whereabnormally low, normal and abnormally high as-AMH directly correlateswith oocytes yields (FIG. 13). Above age 40 years, small patient numbersshould be cause for cautious interpretations. Abnormally low as-AMHstill appears to differentiate oocytes yields from women with abnormallyhigh as-AMH but, considering low overall oocytes yields in this agecategory, differences are no longer pronounced. The most interestingobservation in this age group may, indeed, be that women with normalas-AMH levels appear rare. All here investigated patients in this agegroup were either abnormally low or abnormally high in their respectiveAMH (FIG. 16).

FIG. 13 demonstrates that, among 288 women who reached oocytesretrieval, those with low as-AMH (n=138) yielded at all ages feweroocytes than women with normal as-AMH (n=57) (p<0.05). Above age 30years, those with abnormally high as-AMH (n=92) demonstratedsignificantly higher oocytes yields (p<0.05). Women with abnormally lowas-AMH very clearly differentiated themselves in oocytes numbers fromall other patients (normal and high as-AMH combined) (F=78.9, df 1;p<0.00001).

Discussion of Example 10

Challenges in assessing OR correctly, and limitations of currentlyavailable methodologies have recently been subject to a number ofinsightful publications (Fleming et al. 2006; Sun et al. 2008; Broer etal. 2009; Knauff et al. 2009). Unanimity appears to evolve that AMH inmany ways may represent a more specific marker of DOR than historicallyutilized FSH (Hazout et al., 2004; Ebner et al., 2006; Barad et al.,2009).

However, with few exceptions (Ebner et al. 2006; Gnoth et al. 2008;Barad, et al. 2009; Singer et al. 2009), the literature so far does notoffer cut off values for AMH that may delineate between normal andabnormal OR. Moreover, the literature, with one exception (Barad et al.2009), also so far does not comment on potential differences in utilityof AMH at different female ages, as observed for FSH (Abdalla et al.2004; Toner 2004).

Based on FSH data, we in 2007 suggested that as-ovarian reserveassessments may be superior to nas-testing in predicting DOR andproduction of lower oocytes yields in association with IVF (Barad et al.2007). Sun and associates, who pointed out the importance ofdifferentiating between age-dependent (physiologic) andnon-age-dependent (premature) ovarian aging, recently also suggested asimilar concept (Sun et al. 2008). Considering such evolving concepts,it appeared important to investigate whether as-AMH levels, like as-FSH,may offer improved specificity in detecting DOR over nas-AMH testing.This study did that and, as previously reported for FSH, AMH-datastrongly reemphasize that, judged by oocytes yields in IVF, as-ovarianfunction tests appear superior to nas-testing.

The study population to a large degree represented infertility patientswith significant DOR (FIG. 11). To avoid extremes, we, however, hadeliminated DOR patients with FSH elevations (>12 mIU/ml) in establishingas-95% CIs. The effectiveness of this approach is well documented by thefact that the final study populations still demonstrated the welldescribed declines in AMH (and rises in FSH) with advancing female age((Singer et al. 2009, and FIG. 1).

This study, however, offers important additional insights: Itdemonstrates for the first time that the range of as-AMH is at all agesnarrower than that of as-FSH (FIG. 14). Since narrower testing rangesreflect more specificity, it is not surprising that AMH has been foundto be more specific in reflecting OR than FSH (Barad, et al. 2009).

The figure also demonstrates, however, that both, AMH and FSH,demonstrate the narrowest ranges of as-levels at approximately age 35years. This observation would suggest that at this age both of these ORparameters are probably at their best (i.e., demonstrate highestspecificity) in reflecting ovarian function. Below and above that age,normal ranges widen and, hormone levels, therefore, likely, become lessspecific. This, of course, should not surprise: Abnormally high FSHlevels at younger ages have been reported as less predictable of poortreatment outcomes (Abdalla and Thum 2004; Toner 2004), and wepreviously reported that, though superior to FSH, AMH looses specificityat more advanced female ages (Barad et al. 2009). OR evaluations by FSHand AMH, and even their as-values, therefore, have to be vieweddifferently at different ages.

This study, thus, sheds further light on the value of AMH testing atdifferent ages. As FIG. 16B well demonstrates, even as-AMH, while stillsuperior to nas-AMH, appears to offer its best diagnostic specificityonly at ages 30 to 39 years, correlating well to the narrowest range ofas-AMH at approximately age 35 (FIG. 1). Within that age-range, as-AMHdiscriminates well in regards to oocytes yields at both extremes of AMH:abnormally low levels correlate statistically with abnormally lowoocytes yields, while abnormally high AMH is predictive of high oocyteyields.

While abnormally low oocytes yield is often defined as four or lessretrieved oocytes, such an age-independent definition does not makephysiological sense since expected oocytes yields, of course, are muchhigher at younger than older ages (Singer et al. 2009). In practicalterms this means that four or less oocytes will always represent a lowcount in younger women but may represent an excellent retrieval resultat older age. In the same way, seven or eight oocytes, clearly abovethis widely utilized cut off value, may still represent a low yield in a22-year old. The relativity of oocytes yield, based on female age,therefore, needs to be considered when results of this study areassessed.

At younger ages (<30 years), as-AMH still discriminates risk for lowoocyte yields, but appears insufficiently specific to discriminate highoocytes numbers from normal yields. Because of small patient numbers,this study is limited in its ability to assess the value of as-AMH inwomen above age 40 years. The most interesting finding in this age groupmay, however, be the observation that, at advanced ages, most womenrepresent two specific phenotypes: they either have low or high as-AMH,with few, if any patients, in between. While the small sample size ofpatients in this age group warrants caution in interpreting these data,this finding is potentially interesting since it correlates well withrecent data, developed in DOR patients under dehydroepiandrosterone(DHEA) supplementation. They, largely, either did, or did not, improveAMH levels with DHEA, and occurrence of pregnancy was almost exclusivelylinked to improvements of AMH (Gleicher et al. 2009).

Combined, these data may suggest that, despite declining specificitywith advancing female age, AMH may find a place in defining who, amongstolder women above age 40, may benefit from infertility treatments.

The accurate diagnosis of DOR, therefore, appears important at all ages.This may seem counterintuitive to current clinical practice, whichlargely assumes that younger women, even if afflicted by DOR, stillpossess adequate OR to conceive (Gleicher et al. 2006) and that,therefore, a timely and more accurate diagnosis of DOR in young womenmay be less of a priority than in older women.

While this study does not contradict this argument, accurate and timelydiagnosis in young women may be even more important than in olderpatients since DOR is less clinically obvious at younger ages,frequently unsuspected and overlooked and often leads to aninappropriate diagnosis of so-called unexplained infertility (Gleicheret al. 2006; Barad et al. 2007). Younger women, therefore, may actuallybe the best targets for as-AMH testing. Once suspected of DOR, they thencan be followed closely, and consider time adjustments to familybuilding efforts or fertility preserving treatments.

Comparing here reported differences in as- and nas-AMH to our previouslypublished FSH data (Barad et al. 2007), the discriminatory abilities ofas-AMH in predicting oocytes yields, and therefore insipient DOR, appearsuperior at all ages. These findings, of course, correlate well with thebetter specificity of nas-AMH over nas-FSH (Barad et al. 2009). Whethercombining as-FSH and as-AMH further improves assessments of DOR andexpected oocytes yields is currently under investigation.

These conclusions also correlate well with previously published work byAustrian colleagues: Ebner and associates not only suggested that AMHappears superior to FSH in predicting oocytes numbers and their quality,but actually defined a nas-AMH range for maximal oocytes quality between1.7 and 4.5 ng/mL (Ebner et al. 2006). This range, of course, almostperfectly relates to the normal as-AMH range, defined in this study forwomen up to age 34 years (FIG. 12). It, now, would be interesting todetermine whether this ideal AMH range in regards to egg quality alsochanges with advancing female age or whether the obviously superiorquality of young age is not recoverable at later ages.

Historically, AMH has been primarily utilized to rule out the presenceof DOR. AMH, however, is also potentially useful at the other end of theOR spectrum, when a diagnosis of PCOS is contemplated (Nelson et al.2007; Carlsen et al. 2009; Nelson et al. 2009). AMH cut off values havehere, however, so far also not been defined well, and where attempts atdefinition were made, nas-testing was utilized (Nelson et al. 2009).

While this study does not define diagnostic as-AMH levels for PCOS, itvery clearly demonstrates that the upper 95% CI of as-AMH, at least inwomen at ages 30-39 years, discriminates between normal and abnormallyhigh oocytes yields with IVF (FIG. 13 and FIG. 16B).

Nelson et al suggested that already a nas-AMH of above 15 pmol/L (2.1ng/mL) denotes risk for ovarian hyperstimulation (Nelson et al. 2009).Such an AMH level, on as-basis, is below the lower cut off point inwomen under age 30 years and in the middle of the normal range of womenbetween ages 30 to 34 years (FIG. 12). It, therefore, would include amajority of young women with normal OR, not appear specific enough toidentify patients at risk for ovarian hyperstimulation and clinically beimpractical as a screening tool and rational for changes in stimulationprotocol, as suggested by these authors (Nelson et al. 2009).

In the here reported study 4.6 ng/mL represents the upper limit ofnormal in the youngest, and therefore highest risk, group for ovarianhyperstimulation. While none of the women did develop significantclinical hyperstimulation, some produced excessively high oocytes yields(FIG. 13). More importantly, however, in all age groups above age 30years, the upper 95% CI clearly did define a patient population thatproduces significantly more oocytes than women in normal as-AMH range.

This study, therefore, suggests that as-AMH at all ages allows fordiscrimination of oocytes yields, but does so differently at differentfemale ages. Whether the here utilized methodology of defining normalranges by as-95% CIs represents the best methodology, remains to beseen. The here presented data, however, suggest that as-AMH testingoffers clear advantages over nas-testing and that, whatever ultimate cutoff values shall be chosen to define risk towards abnormal ovarianresponses, they should be age-specific.

By also defining risk towards high oocytes yields, as-AMH thusdemonstrates yet another distinct advantage over as-FSH, which has onlypredictive value for abnormally low oocytes yields (Barad et al. 2007).The prevention of OHSS has, to a degree, remained elusive (Nelson et al.2007), and is especially important in younger women, where risks are thehighest since oocytes production is the largest (Engmann et al. 2008).

Here presented as-AMH ranges, however, need to be viewed with caution:As previously demonstrated for FSH, as-hormone levels will be dependenton study populations (Barad et al. 2007). Since the 95% CI for age willvary dependent on the percentage of women with DOR in each age group, wereported, as one would suspect, different as-FSH levels between IVFcenters, dependent on their respective patient populations. Morefavorably selected patients in one center, therefore, will demonstratelower FSH and higher AMH cut offs than less favorable patients atanother center. In considering the design of this study, we built onabove noted experience and removed from considerations women with veryobvious DOR, defined by FSH levels above 12.0 mIU/mL.

Considering the adverse patient selection and high prevalence ofpremature DOR at our center (FIG. 11, Barad et al., 2007), our patientpopulation may, nevertheless, still be more affected by DOR thanpatients at many other IVF centers. Extrapolating from our previouslypublished FSH data (Barad et al. 2007), it, therefore, seems likely thathere reported as-AMH cut off levels will be conservative for a majorityof infertility centers. Fertility centers with less adversely selectedpatients may, therefore, have to utilize slightly higher as-AMH cut offsat all ages. Preferably, of course, fertility centers should establishcenter-specific as-cut off values until, ideally, universal cut offvalues have been reported for normal, fertile populations, which wouldbe applicable to all women.

Conclusion of Example 10

This study once more demonstrates the advantages of as-OR testing incomparison to currently still widely practiced nas-testing. By havingdemonstrated advantages to as-FSH, and now as-AMH, testing overtraditional nas-testing, it seems increasingly likely that as-testingis, in general, superior to nas-testing when it comes to OR assessments.

Utilizing multiple ovarian reserve parameter, Verhagen and associates,based on a meta-analysis of published studies, recently argued that theuse of more than one OR test can currently not be supported. Afterreviewing multivariate models for prediction of OR and occurrence ofpregnancy with IVF, they concluded that predictive values of variousmodels, utilizing different tests, did not vary significantly from theaccuracy of antral follicle counts as a single test (Verhagen et al.2008).

While the here presented data cannot address the benefits of multiple ORtests over the utilization of only single tests, it is important to notethat Verhagen's meta-analysis involved only nas-testing. It seemspossible, and maybe even likely, that as-testing of OR may givedifferent results.

Like Verhagen et al, we recently compared predictive values for OR andpregnancy, based on receiver operating characteristic (ROC) curves, anddemonstrated a significant advantage of AMH over FSH in predicting bothoutcomes (Barad et al. 2009). Those comparisons, like those of the Dutchinvestigators, were based on nas-testing. By now having demonstratedsignificant advantages of as-over nas-testing for FSH and AMH, it seemsincreasingly likely that only as-use of all OR tests will furtherimprove sensitivity and specificity of such testing. This means that,whether OR testing is performed using FSH, AMH, antral follicle countsor other testing procedures, all should utilize as-, rather than nas-cutoff values.

as-OR tests appear to offer distinct benefits at all ages and may beespecially beneficial for younger women in whom a diagnosis of DOR israrely suspected and, therefore, often overlooked, and who are athighest risk for OHSS.

EXAMPLE 11 Reducing Aneuploidy

Background Overview

Dehydroepiandrosterone (DHEA) has been reported to improve pregnancychances in women with diminished ovarian reserve (DOR), and to reducemiscarriage rates by 50-80%. Such an effect is mathematicallyinconceivable without beneficial effects on embryo ploidy. This study,therefore, assesses effects of DHEA on embryo aneuploidy.

Methods Overview

In a 1:2, matched case control study, 22 consecutive women with DOR,supplemented with DHEA, underwent preimplantation genetic screening(PGS) of embryos during in vitro fertilization (IVF) cycles. Each wasmatched by patient age and time period of IVF with two control IVFcycles without DHEA supplementation (n=44). PGS was performed forchromosomes X, Y, 13, 16, 18, 21 and 22, and involved determination ofnumbers and percentages of aneuploid embryos.

Results Overview

DHEA supplementation to a significant degree reduced number (P=0.029)and percentages (P<0.001) of aneuploid embryos, adjusted for relevantcovariates. Short term supplementation (4-12 weeks) resulted in greatestreduction in aneuploidy (21.6%, 95% CI-2.871-46.031).

Discussion Overview

Beneficial DHEA effects on DOR patients, at least partially, are thelikely consequence of lower embryo aneuploidy. DHEA supplementation alsodeserves investigation in older fertile women, attempting to conceive,where a similar effect, potentially, could positively affect publichealth.

Methods of Example 11

Patient Populations

We retrieved from our center's computerized research data bank a totalof 22 consecutive DOR patients who underwent IVF/PGS while on DHEAsupplementation. Only first IVF cycles were analyzed. These cycles werematched with the two control cycles not on DHEA supplementation, basedon patient age and year of treatment (44 controls).

A diagnosis of DOR was reached if patients demonstrated abnormallyelevated age-specific baseline follicle stimulating hormone (FSH) orabnormally low age-specific anti-Müllerian hormone (AMH) levels. Normalage-specific hormone levels were defined by 95% confidence intervals atall ages, as previously reported. Since patients with DOR were, thus,uniformly diagnosed before IVF cycle protocols were determined, theywere all supplemented with DHEA, and stimulation adjustments were made.This results in improved oocyte and embryo yields in comparison topatients who are not diagnosed with use of age-specific FSH and AMH.

DHEA Supplementation

During the study period all DOR patients at our center routinelyreceived DHEA supplementation. Those not receiving DHEA, therefore, bydefinition, had age-appropriate ovarian reserve, confirmed by normalanti-Müllerian hormone (AMH), follicle stimulating hormone (FSH)baseline levels and estradiol. Patients receiving DHEA supplementationwere prescribed 25 mg of micronized, pharmaceutical grade DHEA, T.I.D,for at least four weeks prior to IVF cycle start. Short-termsupplementation was defined as 4 to 12 weeks of DHEA prior to IVF andPGS; supplementation beyond that was considered long-termsupplementation.

Ovarian Stimulation

Patients with premature DOR, or if over 40 years of age, in firsttreatment cycles universally receive a so-called microdose gonadotropinreleasing hormone agonist (GnRH-a) ovarian stimulation protocol,characterized by leuprolide acetate (50 μg/0.1 mL, b.i.d.; Lupron®,Abbot Pharmaceuticals, North Chicago, Ill.) and ovarian stimulation withfollicle stimulating hormone (FSH, 300 IU-450 IU daily) and humanmenopausal gonadotropins (hMG, 150 IU). If under age 40 with normalovarian reserve patients receive down regulation with full dose GnRH-a(1.0 mg/0.1 mL) and ovarian stimulation with up to 300 IU ofgonadotropins, usually half as FSH and half as hMG. Higher luteinizinghormone (LH) contributions to ovarian stimulation, if anything, reduceembryo aneuploidy. The small difference in ovarian stimulation protocolsbetween women with DOR and controls, therefore, potentially biases studyoutcome towards lower aneuploidy rates in the control population, whichreceived a proportionally higher LH contributions to ovarianstimulation.

Preimplantation Genetic Screening (PGS)

PGS was performed, utilizing fluorescence in situ hybridization (FISH)in routine fashion, utilizing probes for seven chromosomes (X, Y, 13,16, 18, 21 and 22) on day three after fertilization, when embryosreached six to eight cell stages. This restricted chromosome panel iscurrently routinely utilized for PGS.

Statistical Analysis

A general linear model was constructed to assess DHEA effects on percentaneuploidy after adjustment for age, indications for PGS, stimulationprotocol and total gonadotropin dosage utilized. The latter adjustmentwas made as a surrogate for potential physician biases in how individualpatients were stimulated, and potential effects such stimulation biasesmay have on ploidy.

All patients at our center sign a universal informed consent at time ofinitial presentation, which permits the extraction of clinical data frompatient records as long as confidentiality of the record and anonymityof patients is maintained. The center's Institutional Review Board,therefore, permits such studies under expedited review.

Results of Example 11

Patients

Table 14 showing patient characteristics summarizes characteristics ofstudy and control patients. The two groups did not differ in age andrace/ethnicity. DHEA patients were significantly more obese butexpressed poorer ovarian reserve, based on lower AMH (P=0.045) andsignificantly higher gonadotropin utilization (P=0.002). Such aconclusion was also supported by trends towards higher FSH and smalleroocyte yields (9.6±6.2 vs. 11.7±6.3). Embryo numbers transferred(1.4±0.9 vs. 1.5±0.7), embryos cryopreserved (0.7±1.6 vs. 0.6±1.2),embryos undergoing PGS (7.3±3.9 vs. 6.6±3.6) and embryo grades (3.4±0.4vs. 3.5±0.3) were similar between both groups.

TABLE 14 Patient characteristics Patient characteristics DHEA ControlsP-value Number 22 44 Age (years, Mean ± SD) 37.9 ± 4.7  37.2 ± 4.4  N.S.Race/ethnicity (%) Caucasian 14 (63.6) 21 (47.7) African 2 (9.1) 3 (6.8)Asian 4 (18.2) 14 (31.8) Middle Eastern 1 (4.5) 6 (13.6) BMI (Mean ± SD)24.4 ± 3.8  21.0 ± 1.7  0.006 AMH (ng/mL, mean ± SD) 1.3 ± 1.2 2.0 ± 1.90.045 Range 0.8-2.1 1.0-4.4 FSH (mIU/mL, mean ± SD) 10.1 ± 6.8  8.0 ±5.5 N.S. Range  8.4-12.2 7.1-9.1 Oocytes retrieved 9.6 ± 6.2 11.7 ± 6.3 N.S. (n, mean ± SD) Embryos (n, mean ± SD) Transferred 1.4 ± 0.9 1.5 ±0.7 Cryopreserved 0.7 ± 1.6 0.6 ± 1.2 Undergoing PGS 7.3 ± 3.9 6.6 ± 3.6Grades 3.4 ± 0.4 3.5 ± 0.3 Total gonadotropins 5711 ± 1818 4048 ± 18860.002 dosage (IU, mean ± SD) ¹ Difference in BMI was based onsignificant difference in weight (P = 0.02) not height (P = 0.24)

Aneuploidy

As demonstrated in FIGS. 25A and 25B, a comparison of absolute andpercentages of aneuploidy in DHEA and control patients, demonstrate thataneuploid embryos were significantly more prevalent amongst controls(4.5±3.1 vs. 2.8±2.5; P=0.03), as were percentages of aneuploidy(61.0±22.4 vs. 38.2±24.4; P<0.001). In the general linear model, afteradjustment for age, FSH dose and indication for PGS, the association ofDHEA supplementation effects on ploidy remained significant (F=13.2, df1, p=0.001). As expected, women who underwent PGS for aneuploidyscreening had a greater percentage of aneuploidy embryos than women whounderwent PGS for elective gender selection purposes (P<0.007).

Possibly because of still relatively small study numbers, no specificaneuploidy pattern, affecting distinct chromosomes, was apparent.

Mean length of DHEA supplementation was 7.3±2.2 weeks in the short and19.1±9.1 weeks in the long treatment group. Women in the short treatmentgroup demonstrated the greatest reduction in aneuploidy (21.6%, 95%CI-2.871-46.031).

Discussion of Example 11

This study supports prior preliminary evidence that DHEA supplementationreduces aneuploidy in women with DOR, first suggested in a small pilotstudy, when at least one euploid embryo was found significantly morefrequently after DHEA than in matched control cycles. Subsequently, wealso demonstrated that DHEA supplementation reduces miscarriage rates toa degree that cannot be explained without significant contribution fromreduced aneuploidy.

By demonstrating no difference in embryo grades between DHEA and controlcycles (Table 14), this study also demonstrates, once more, that embryomorphology, as currently routinely assessed in most IVF laboratories,does not reflect on embryo ploidy and, therefore, is limited in clinicalvalue.

Since a majority of miscarriages are believed to be consequence ofaneuploidy, decreases in aneuploidy rate should translate into decreasesin spontaneous pregnancy loss. Two infertility centers utilizing DHEAsupplementation, one in New York City and the other in Toronto, Canada,indeed, independently, reported identically low miscarriage rates of15.0 and 15.2 percent, respectively. Depending on method of statisticalanalysis, these miscarriage rates represented declines of approximately50 to 80 percent from expectations. Even more remarkably, the combinedloss rate of 15.1 percent equated rates reported for normal populationsas young as 28 to 33 years, and was, thus, far removed from excessivelyhigh miscarriage rates, reported in DOR patients.

Even though such significant declines in spontaneous miscarriages cannotbe achieved without underlying improvements in aneuploidy, miscarriagerates only represent surrogates for true aneuploidy studies. Directevidence for such an effect was, therefore, still needed.

In this study we for the first time are able to demonstrate such directevidence, utilizing routinely performed PGS of preimplantation stageembryos, performed in DHEA supplemented IVF cycles and controls. DHEAsupplementation was, indeed, associated with significantly reducedaneuploidy, and greatest reductions were observed with short DHEAsupplementation of up to 12 weeks.

This observation on first impulse suggests that, excluding month one ofsupplementation, second and third months offer the best chance oflowering aneuploidy, thus fully supporting previously publishedpregnancy data after DHEA supplementation, which demonstrated asignificant first rise in pregnancy rate after approximately six weeksof DHEA supplementation. Six weeks of DHEA supplementation prior to IVFcycle start, therefore, currently represents minimal supplementationtime at our center.

This study, however, does not preclude, as alternative explanation forthese findings that a more favorable patient group conceives quicklyand, therefore, statistically distorts above noted time associations.Such a possibility cannot be ruled out since we previously demonstratedthat women who improve AMH levels with DHEA supplementation demonstratesignificantly superior pregnancy rates to those who do not.

A general criticism of currently available technologies for PGS is thatonly limited numbers of chromosomes can be evaluated (24 chromosomescreening technologies are currently under investigation). In thisstudy, this meant that only seven chromosomes were assessed in study andcontrol patients. This allows for the at least theoretical possibilitythat untested chromosomes demonstrate statistically different aneuploidydistributions from the here tested seven and that, including a fullchromosome complement, here reported differences would disappear. Suchan explanation is, however, highly unlikely, and the here utilizedselection of chromosomes, or similar ones, have been routinely used inclinical PGS. There is also no data in either human or animal literatureto suggest that DOR maybe associated with aneuploidy of specificchromosomes.

Because of time pressures, when using their own oocytes, prospectivelyrandomized clinical trials in patient populations affected by DOR, andinvolving placebo, are difficult, if not impossible, to conduct. Ourcenter, for that reason, had to abandon two registered clinical DHEAtrials, one in the United States and one in Europe, due to lack ofenrollment. A small first such trial has just been reported. Bestavailable evidence, therefore, at least in part, has to be obtained viaother study formats.

In this study, the format chosen was a case control study in which eachstudy patient/cycle was matched with two controls. As Table 14demonstrates, patient and control populations appear, with fewexceptions, overall comparable. It is, however, important to point outthat the significantly larger preponderance of DOR in the study group(Table 14) biases study results against discovery of DHEA effects onploidy since DOR patients demonstrate the highest aneuploidy rateamongst infertility patients. Even just absence of increased aneuploidyin the study group could, therefore, be viewed as a potentially positiveDHEA effect. Instead, this study actually demonstrates significantlylower aneuploidy following DHEA supplementation.

How DHEA affects non-dysfunctional events remains to be determined butwe have speculated that DHEA supplementation may improve the ovarianenvironment in which follicular maturation takes place in older women.DHEA, indeed, significantly declines with advancing age. Since DHEA,except in our prior pilot study, has never before been directlyassociated with decreases in aneuploidy, neither animal nor human dataare currently available to speculate further on specific mechanisms thatmay be involved.

Others have speculated that drugs can be developed which beneficiallyaffect non-dysjunctional events during meiosis. DHEA may, indeed, turnout to be a first pharmacologic agent to do so.

This effect, only unlikely, should be restricted to infertile women withDOR. DHEA supplementation, in attempts to reduce embryo aneuploidy andspontaneous miscarriages, therefore, also deserves investigation in,especially older (above age 35 years) fertile women, attemptingconception. A possible similar beneficial impact in fertile patientpopulations, attempting spontaneous conception, could have a majorimpact on public health by speeding up time to pregnancy and by reducingembryo aneuploidy and miscarriage rates.

EXAMPLE 12 Improvement in Diminished Ovarian Reserve

Dehydroepiandrosterone (DHEA) has been reported to improve oocyte/embryoyields and oocyte/embryo quality in women with diminished ovarianreserve. In this study, we show that DHEA objectively improves ovarianreserve. This study investigated 120 consecutive patients withdiminished ovarian reserve, supplemented for about 30- about 120 days(mean 73±27) with DHEA (about 25 mg three times daily). Anti-Müllerianhormone (AMH) concentrations were determined relationship to DHEAsupplementation using linear regression and, longitudinally, byexamining interaction between days of DHEA treatment and pregnancysuccess in respect to changes in AMH. AMH concentrations significantlyimproved after DHEA supplementation over time (P=0.002). Women underabout age 38 years demonstrated higher AMH concentrations and improvedAMH concentrations more than older females. AMH improved longitudinallyby approximately 60% (P<0.0002). Women reaching IVF experienced about a23.64% clinical pregnancy rate and conceiving women showed significantlyimproved AMH concentrations compared with those who did not (P=0.001).DHEA supplementation, thus, significantly improved ovarian reserve inparallel with longer DHEA use and was more pronounced in younger women.

Materials and Methods Of Example 12

The study is a cross-sectional and longitudinal analysis of 120consecutive women with diminished ovarian reserve, in whom AMHconcentrations were evaluated as a reflection of ovarian reserve. FirstAMH concentrations obtained were used for initial analysis. Post DHEAinitiation, exposure to the supplement ranged from 34 to 119 days (mean73±27 days). Women with two or more consecutive AMH concentrationscomprised patients in the longitudinal study evaluation of ovarianreserve after initiation of DHEA supplementation.

The study center defines diminished ovarian reserve in women under age40 by elevated age-specific FSH concentrations (on cycle days 2 or 3) orby universal AMH concentrations (obtained on a random day of themenstrual cycle) below 0.8 ng/ml, which approximately correlates to anFSH of 11.0 mIU/ml. Since ovarian reserve declines with advancing femaleage, women above age 40 are uniformly assumed to suffer from diminishedovarian reserve. Age-specific FSH concentrations have, in associationwith IVF, been demonstrated to discriminate between oocyte yields.Age-specific FSH cut offs were as follows: <7.0 mIU/ml under age 33;<7.9 mIU/ml ages 33-37 years; <8.4 mIU/ml ages 38-40; <8.5 mIU/ml at orabove age 41 years.

FSH and oestradiol were evaluated by standard enzyme-linkedimmunoabsorbent assay (ELISA; AIA-600II, Tohso, Tokyo, Japan). Onlyresults in assay range were considered for statistical evaluation. AMHconcentrations were also obtained by ELISA. In short, the DSL-14400active Müllerian Inhibiting Substance/Anti-Müllerian Hormone (MIS/AMH)ELISA was utilized (Diagnostic Systems Laboratories, Webster, USA). Thisis an enzymatically amplified two-site immunoassay, which does not crossreact with other members of the transforming growth factor superfamily,including TGFβ1, BMP4 and ACT. Theoretical sensitivity, or minimumdetection limit, as calculated by interpolation of the mean plus two SDof eight replicates of the 0 ng/ml MIS/AMH standard, is 0.006 ng/ml.Intra-assay coefficient of variation for an overall average AMHconcentration is ≦20%.

Since 2007, diminished ovarian reserve patients at the study centre havereceived for at least 2 months supplementation withpharmaceutical-grade, micronized and pharmacy-compounded DHEA at dosagesof 25 mg three times daily before progressing to IVF. DHEA is continueduninterrupted until conception (second, normally rising positivepregnancy test) or until patients discontinue treatment with autologousoocytes.

The study population was age stratified to under 38 years and age 38years and over, and further stratified based on whether clinicalpregnancy had been achieved or not. Age 38 was chosen as cut-off becauseit has been reported to represents the beginning of accelerated declinein ovarian reserve.

Data are shown as mean±SD or as raw numbers and percentages. Data thatwere not normally distributed were log-transformed to enable use ofparametric tests. Data analysis was performed using Statistical Packagefor Social Sciences for Windows version 17.0 (SPSS, USA). Demographicand biochemical data were analyzed with paired or unpaired Student'st-test. A generalized linear model was performed to evaluate theinteraction of pregnancy status with days of DHEA exposure, adjusted forage at start of treatment.

Results of Example 12

The patient population comprised about 74% Caucasians, about 11%African-American and about 15% Asian patients. A large majority (85%)were recorded with a primary diagnosis of diminished ovarian reserve, 3%with male factor infertility and 12% with tubal factor.

Table 15 summarizes the characteristics of the study populations,separately for cross-sectional (n=120) and longitudinal assessments(n=55). Obviously, low baseline AMH and high FSH concentrations areconfirmatory of significant diminished ovarian reserve in the studypopulation. Age ranges also confirm that the younger patient population,indeed, does reflect relatively young infertility patients and,therefore, with a considerable prevalence of premature ovarian ageing,while the older age group in principle represents women above age 38,although a very large majority women above age 40 years.

TABLE 15 Characteristics of study patients. Cross- sectionalLongitudinal study group Parameter study group All <38 years ≧38 yearsP-value No. of patients 120 55 18 37 Age (years)  39 ± 3.9  39 ± 3.134.9 ± 3.1  42.1 ± 1.2  <0.001 AMH (ng/ml) 0.32 ± 0.20 0.22 ± 0.22 0.20± 0.16 0.23 ± 0.17 NS Baseline FSH 15.9 ± 14.1 15.4 ± 9.1  14.2 ± 8.2 18.0 ± 10.8 NS (mIU/ml) Oestradiol (pg/ml) 60.0 ± 50.0 52.3 ± 28.6 56.1± 13.6 53.2 ± 36.6 NS Maximal DHEA-S 474 ± 145 476 ± 180 475 ± 224 478 ±180 NS (μg/dl)^(a) Values are mean ± SD unless otherwise stated. DHEA-S= dehydroepiandrosterone supplementation; NS = not significant.^(a)First follow-up P-value obtained 30 days after initiation of DHEAsupplementation.

Cross-sectional evaluation of the whole patient population (FIG. 26)demonstrates, unadjusted for age, AMH concentrations as a function oflength of DHEA supplementation. The figure demonstrates a steadyincrease in AMH over time until 120 days after initiation of DHEA(P=0.002). Age (P=0.007) and length of DHEA supplementation (P=0.019)were independently associated with AMH concentrations. Younger women,under age 38 years, demonstrated higher AMH concentrations from baselineand proportionally improved AMH concentrations over time afterinitiation of DHEA more than older women at, or above, 38 years.

FIG. 26 is a cross-sectional evaluation of anti-Müllerian hormone (AMH)concentrations in correlation to time from dehydroepiandrosterone (DHEA)initiation. The data are age stratified for under age 38 and ≧38 years.AMH concentrations improved over time significantly for the whole group(P=0.002). Age (P=0.007) and length of treatment (P=0.019) wereindependently associated with increasing AMH concentrations. Fouroutliers outside of range are not shown. This figure representscross-sectional data, with every patient being represented only once, attime of first assessment.

Very similar results were obtained in longitudinal evaluation: Here, AMHconcentrations improved from 0.22±0.22 ng/ml at baseline, before startof DHEA, to 0.35±0.03 ng/ml at highest measured peak, an almost 60%improvement in mean (P=0.0001). FIG. 27 graphically demonstrates thelongitudinal follow up between first and second AMH concentration inindividual patients. The footnote also demonstrates the severity ofdiminished ovarian reserve in these patients, based on AMH and FSHconcentrations.

FIG. 27 shows the anti-Müllerian hormone (AMH) concentrations inindividual patients in longitudinal follow up. This figure summarizesthe progression in individual AMH concentrations between initial andfollow-up evaluations. Mean±SD follow-up time was 155.8±119.1 days; meanAMH was 0.31 ng/ml; mean FSH was 18.0±17.2 mIU/ml. All shown patientsstarted with AMH<0.8 ng/ml.

Amongst 55 women who had undergone IVF, by time of analysis, 13 (23.64%)conceived a clinical pregnancy. FIG. 28 demonstrates a retroactivecomparison of AMH concentrations after DHEA supplementation, in womenwho did and did not conceive. As the figure demonstrates, those whoconceived demonstrated a significantly better AMH response followingDHEA supplementation than unsuccessful patients, whose AMH responseremained flat (interaction of pregnant versus non-pregnant, Waldchi-squared 11.6; df=1; P=0.001).

FIG. 28 shows anti-Müllerian hormone (AMH) concentrations over time fromDHEA initiation in women who did and did not conceive. The data arestratified by pregnancy achieved or not achieved. Interaction ofpregnant versus non-pregnant, Wald chi-squared test 11.6; df=1; P=0.001.

Discussion of Example 12

By assessing changes in AMH concentrations, this study for the firsttime presents objective evidence that DHEA supplementation positivelyaffects diminished ovarian reserve. This DHEA effect is visible inyounger and older ovaries (FIG. 26), but is more pronounced in youngerwomen with premature ovarian ageing.

This study also strongly suggests that observed improvements in ovarianreserve after DHEA supplementation lead to better pregnancy rates. AsTable 15 demonstrates, AMH and FSH concentrations in the studypopulation are highly confirmatory of a significant degree of diminishedovarian reserve.

Even in an adversely selected patient population, approximately one infour women still conceived with use of autologous oocytes is remarkable.It is, however, especially remarkable that, as FIG. 28 demonstrates, theovarian response pattern to DHEA is so dramatically different betweenthose women who ended up conceiving and those who did not. While thosewith future pregnancies demonstrated remarkable improvements in AMHconcentrations, unsuccessful women demonstrated no response whatsoeverto DHEA. They, thus, for practical purposes can be seen as a controlgroup: where DHEA does not improve ovarian reserve, as indicated by flatAMH concentrations, pregnancy is very unlikely.

At least part of DHEA's effect may be a reduction in oocytes and embryoaneuploidy. This study, however, for the first time offers a more directand clinically practical explanation for DHEA effects in women withdiminished ovarian reserve.

The concept of ovarian reserve previously has been based on a presumedremaining follicular pool within ovaries. As this pool shrinks, ovarianreserve, and with it female fecundity, declines. In the process, thesize of immature follicular cohorts, recruited each month, also declinesand as cohorts decline in size, smaller and smaller follicle numbersreach gonadotrophin sensitivity, the last stage of follicularmaturation. As a consequence, follicle numbers and oocytes yield in IVFdecline with advancing female age, as does female fecundity in general.

In fertility practice, follicle numbers and oocyte yield are consideredultimate measures of ovarian reserve. Indeed, AMH is increasinglyconsidered a better reflection of ovarian reserve because it betterpredicts oocyte yield in IVF than FSH. AMH, a dimeric glycoprotein and amember of the transforming growth factor superfamily, is exclusivelyproduced by granulose cells of early developing follicles, from primaryto antral follicle. AMH is, thus, reflective of small, pre-antralfollicles but not of the later stage follicular pool, better representedby FSH concentrations. AMH appears to better reflect total quantity and,possibly, quality of the remaining follicular pool, and, therefore, tobe a better marker of declining reproductive age, an observation whichpotentially explains how DHEA affects ovarian function.

By demonstrating improving AMH concentrations, this study suggests thatin selected patients with diminished ovarian reserve, DHEA progressivelyimproves ovarian reserve at follicular stages at which AMH is produced.This means that over time, DHEA increases the pool of follicles up topre-antral stage, causing a steady improvement in AMH up to 120 dayspost DHEA initiation, as shown here.

DHEA may positively affect recruitment suggesting progressively morepre-antral follicles accumulating and/or follicle quality improvingoverall, resulting in the here-documented increase in AMH over time frominitiation of DHEA supplementation.

Women with very severe diminished ovarian reserve (AMH concentrationsranging from undetectable to 0.4 ng/ml), who after DHEA supplementationachieved pregnancy and demonstrate surprisingly low spontaneouspregnancy wastage, increasingly appear to point towards a DHEA effect onthe ovarian environment within which folliculogenesis takes place. Undersuch a concept, DHEA may represent a first medication which, byimproving the ovarian environment, returns it to a ‘younger’functionality, thus explaining better egg and embryo quality, higherpregnancy and lower miscarriage rates.

Even longer-term therapy than utilized in this study, in similardosages, is safe. In the present study centre's experience, the mostfrequent side effect of DHEA supplementation has been a positive one:women feel energized and report improved sex drives. Adverse sideeffects have been limited to oily skin, rarely mild acne vulgaris andmild hair loss. Since first-trimester placenta produces DHEA, maternalDHEA exposure at moderate dosages in very early pregnancy should notconstitute significant risk.

FIG. 28 suggests that beneficial effects of DHEA may become apparentearlier than two months and may be reflected in spontaneous pregnancies.

While improving AMH concentrations in women with diminished ovarianreserve appear closely associated with pregnancy success, AMH may not besensitive enough to predict who will or will not conceive. Pregnanciescan even be established at undetectable AMH concentrations. This meansthat AMH concentrations alone will not allow discrimination between whodoes and does not deserve further infertility treatments.

As this study, however, demonstrates, AMH offers objective evidence forthe therapeutic efficacy of DHEA in women with diminished ovarianreserve and especially under about age 38 years. Moreover, a good AMHresponse to DHEA supplementation clearly discriminates between good andpoor prognosis patients in regards to pregnancy success. Thisinformation alone will improve patient counseling in women withsignificant diminished ovarian reserve. Other markers of ovarian reserveare being investigated currently in attempts to even better predictsuccess of DHEA supplementation and, thus, avoid such treatment in womenwho will not have improved pregnancy chances in response to DHEAsupplementation.

EXAMPLE 13 AMH and Live-Birth Chances

Overview of Example 13

Maximal receiver operating characteristic curve inflections, whichdifferentiate between better and poorer delivery chances in women withdiminished ovarian reserve (DOR) independent of age, were atanti-Müllerian hormone (AMH) 1.05 ng/mL (improved odds for live birth4.6 [2.3-9.1), 95% confidence interval; Wald 18.8, df=1], although livebirths occurred even with undetectable AMH. Pregnancy wastage was verylow at AMH≦0.04 ng/mL but significantly increased at AMH 0.41-1.05ng/mL, resulting in similarly low live-birth rates at all AMHlevels≦1.05 ng/mL and significantly improved live-birth rates atAMH≧1.06 ng/mL.

Details of Example 13

Diminished ovarian reserve (DOR) predicts pregnancy chances. Youngerwomen usually do better. DOR is associated with pregnancy loss,resulting in disappointing live-birth rates. Which DOR patients maybenefit from treatment is, therefore, potentially important. Currentovarian reserve assessments do not allow distinction. A suitable testwould, therefore, be welcome. Anti-Müllerian hormone (AMH) betterpredicts DOR than FSH.

Whether an AMH value discriminates poorer from better live-birthchances, is, however, unknown. FSH has been unable to do so. AMH'sspecificity decreases as women age and/or develop DOR.

Pregnancies at undetectable and undetectable to low (≦0.4 ng/mL) AMHconfirm this. Ultimately important is, however, whether AMH can predictlive births.

295 DOR patients with AMH evaluations reached IVF (507 cycles). DOR wasinitially defined by FSH above 10.0 mIU/mL and/or ovarian resistance tostimulation (four or fewer oocytes). Age-specific FSH and age-specificAMH levels were established, which defined DOR by abnormally highage-specific FSH and/or abnormally low age-specific AMH.

One purpose of this study was to determine an AMH cutoff value thatdiscriminates between better and poorer live-birth chances. This wasdone using receiver operating characteristic (ROC) curves for the wholepopulation and, separately, for different age categories. A maximalinflection point between lower and higher live birth chances wasuniformly (independent of age) an AMH level of 1.05 ng/mL. See at leastFIG. 29.

In FIG. 29 shows a ROC curve of AMH and live births involving 507 IVFcycles in 295 women with DOR. The star indicates point of maximalinflection, representing, as the set in table 16 demonstrates, an AMHvalue of 1.05 ng/mL. Not shown here are ROC curves at ages 30-35, 36-40,and >40 years, all demonstrating the same point of maximal inflectionbetween lower and higher live births. The value of 1.05 ng/mL thusrepresents a uniform cutoff between lower and higher live-birth chance,independent of age.

AMH<1.05 ng/mL represents more severe DOR. The study encompassed 174severe (310 IVF cycles) and 121 milder (183 IVF cycles) DOR patients,with characteristics shown in Table 16. Chances of clinical pregnancies,miscarriages, terminations of pregnancy, and viable deliveries were thendetermined among severe DOR patients, depending on AMH levels (belowdetection, <0.1 ng/mL, 0.1-0.4 ng/mL, 0.41-0.8 ng/mL, 0.81-1.05 ng/mL)and with milder DOR (>1.06 ng/mL). Our routine protocol for DORsupplements patients with dehydroepiandrosterone (DHEA) and stimulateswith microdose agonist cycles.

TABLE 16 Patient characteristics. AMH ≦ 1.05 AMH > 1.05 Patientcharacteristics ng/mL (n = 174) ng/mL (n = 121) Age 39.2 ± 4.6 35.2 ±5.4^(a) AMH, ng/mL 0.44 ± 0.3  2.6 ± 1.8^(a) BMI 25.7 ± 6.4 23.6 ±6.2^(b) E₂, pg/mL  45.8 ± 20.3  48.1 ± 23.2 FSH, mIU/mL  20.2 ± 18.710.8 ± 9.8^(a) Months in treatment  3.9 ± 3.7  4.2 ± 4.1 Race, n (%):Caucasian 125 (71.8) 79 (65.3) African American 17 (9.8) 16 (13.2) Asian32 (18.4) 26 (21.5) Primary infertility diagnoses, n (%): DOR 102 (58.6)36 (29.8)^(a) Endometriosis 8 (4.6) 2 (1.7) Male factor 39 (22.4) 35(28.9) PCOS 1 (0.6) 6 (5.0)^(c) Tubal infertility 7 (4.0) 19 (15.7)^(b)Uterine pathology 0 (0.0) 1 (0.8) Other 17 (9.8) 22 (18.2)^(c) ^(a)P <.001; ^(b)P < .01; ^(c)P < .05

Data are mean±SD or n (%). Table 16 demonstrates that women withAMH≦1.05 ng/mL (severe DOR) are older, have lower AMH, higher FSH, andhigher body mass index (BMI) but do not differ in E2 levels and lengthof treatments. They also represent a significantly higher prevalence ofDOR as primary infertility diagnosis and fewer cases of PCOS, tubalfactor infertility, and other diagnoses.

AMH levels were obtained, using the DSL-10-14400 active Müllerianinhibiting substance/AMH (MIS/AMH) enzyme-linked immunoabsorbent assay(Diagnostic Systems Laboratories, Webster, Tex.) (17). The theoreticalsensitivity or minimum detection limit is 0.006 ng/mL. The inter- andintra-assay coefficient of variation reported by manufacturer is <10%and was <15% here.

Data are shown as means±standard deviation (SD) or raw numbers andpercentages. Normally distributed data were compared by one-way analysisof variance, categorical data by χ2. Live births were assessed usinglogistic regression. Logistic regression was performed with live birthas the dependent variable and AMH≦ or >1.05 ng/mL, adjusted for age,months in treatment, diagnosis, and race.

Data analysis was performed using SPSS for windows, version 17.0 (SPSSInc., Chicago). Differences were considered to be statisticallysignificant if P<0.05.

Table 16 demonstrates that patients with AMH≦1.05 ng/mL were older(39.2±4.6 years vs. 35.2±5.4 years; P<0.001), demonstrated lower AMH(P<0.001) and higher FSH (P<0.001), had higher body mass index (P<0.01),more DOR as admission diagnosis (58.6% vs. 29.8%; P<0.001), lesspolycystic ovarian syndrome (PCOS; P<0.05), and less tubal disease(P<0.01).

FIGS. 30A and 30B demonstrate pregnancy rates per IVF cycle in the upperpanel and cumulative pregnancy rates (independent of length oftreatment) in the lower panel. Table 16 demonstrates, however, thatlength of treatment did not differ below and above AMH 1.05 ng/mL. Thefigures demonstrate that clinical pregnancies can be established at allAMH levels—even in the absence of detectable AMH. Pregnancy ratesremain, however, low (˜5.0 percent per IVF cycle and 10.0% cumulatively)up to AMH 0.4 ng/mL.

FIGS. 30A and 30B demonstrate at various AMH levels percentages of livebirths (LB), terminations of pregnancy for aneuploidy (TOP), andspontaneous miscarriages (SAB) per IVF cycle (upper panel) andcumulatively over length of infertility treatment with IVF (lowerpanel). For further details, see text.

Rates then increase at AMH 0.41-1.05 ng/mL to approximately 10.0% percycle/15.0% cumulatively and significantly improve further above AMH1.06 ng/mL (approximately 25.0%/cycle, 40.0% cumulatively; P<0.001).Among 507 IVF cycles, 320 were in women with severe DOR and 24 (7.5%)led to clinical pregnancy, while among 136 milder DOR patients, 51clinical pregnancies were established (37.5%), which is a significantlyhigher pregnancy rate (χ2, 62.5, df=1, P<0.0001).

Live-birth rates differ significantly from clinical pregnancy rates(FIGS. 30A and 30B) because of higher wastage at AMH 0.41-1.05 ng/mL(P<0.05), leading to loss of the previously observed statisticaladvantage in clinical pregnancies at AMH 0.41-1.05 ng/mL in comparisonwith lower AMH. Consequently, all AMH categories under 1.05 ng/mLdemonstrate statistically similar low live-birth rates. In logisticregression for the whole study population, with live births as thedependent variable and AMH≦1.05/>1.05 ng/mL, adjusted for age, length ofinfertility treatment, diagnosis, and race, models for all two-wayinteractions were not significant and neither was a Hosmer-Lemeshow testof goodness of fit.

Only AMH (P=0.001) and age (P<0.0001) were significantly associated withthe occurrence of live births. The final model included AMH with age andlength of infertility treatment (in months) as covariates: The oddsratio for live births in the presence of an initial AMH>1.05 ng/mL,adjusted for age and length of treatment, was 4.6 (95% confidenceinterval [2.3-9.2], Wald 18.8, df=1, P<0.001).

Correct assessment of ovarian reserve (OR) is crucial. AMH may offerimproved specificity in predicting ovarian response and pregnancychances. Consequently, AMH has been asserting increasing primacy. AMHmay be used to predict pregnancy and now may predict live births.

Pregnancies and live births do not necessarily run in parallel.Significantly higher miscarriage rates may occur in DOR women than innormal OR patients. Patients with more severe DOR, therefore, mayexperience higher miscarriage and lower live-birth rates.

AMH levels decline and DOR increases with advancing female age, which isalso associated with increasing aneuploidy and miscarriage rates.Increasing miscarriage rates will result in lower live-birth rates and,in turn, be associated with declining AMH levels. Female age and DOR,independently, should therefore be associated with decreasing live-birthrates, as here confirmed, since, among all patient characteristics, onlyage and OR (per AMH) were statistically associated with live births.Birth rates should, therefore, decline in parallel with declining AMH.Concentrating on pregnancy rates may, therefore, be misleading indirecting patient advice. For example, patients with severe DOR maystill demonstrate reasonable pregnancy, although they may demonstrateunacceptably low live-birth rates due to high pregnancy wastage.

This study, however, does not support such a parallel decline inlive-birth rates and AMH. Surprisingly, pregnancy wastage appearsunusually low at the lowest AMH levels, including a complete absence ofdetectable AMH, peaks at midrange (AMH 0.41-1.05 ng/m/L), and fallsagain at AMH>1.05 ng/mL (FIG. 1).

Any advantage in clinical pregnancies between AMH 0.41-1.05 ng/mL andlower levels disappear by delivery, and live-birth rates betweenundetectable and AMH levels of 1.05 ng/mL are statisticallyindistinguishably low. Since live births increase significantly aboveAMH 1.05 ng/mL, it should not be a surprise that an AMH level of 1.05ng/mL, at all ages, represents maximal inflection on ROC curves,differentiating between lower and higher live-birth chances. As the onlyAMH cutoff established with live births, it therefore likely representsthe most reliable definition of severe DOR.

It also almost perfectly correlates to FSH 10 mIU/mL, while an AMH of0.8 ng/mL would approximately correlate to an FSH of 11.0 mIU/mL 12, 24.Clinically, these distinctions are important because, especially inyounger women, FSH levels at or above 10 mIU/mL result in excellentpregnancy chances. Since women with premature ovarian senescence do notdemonstrate increased embryo aneuploidy, such patients should experienceonly minor pregnancy wastage and satisfactory live births. This study,however, suggests that this will be the case only at AMH>1.05 ng/mL.

AMH<1.05 ng/mL, however, does not define DOR. It only defines DOR withsignificantly decreased live-birth chances. It also does not warrantwithholding of treatment because even DOR patients with very low toundetectable AMH still achieve rather surprising live-birth rates.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific exemplary embodiments thereof. The invention is thereforeto be limited not by the exemplary embodiments herein, but by allembodiments within the scope and spirit of the appended claims.

What is claimed is:
 1. A method of reducing aneuploidy rates in humanembryos, said method comprising: administering an androgen selected fromthe group consisting of DHEA and testosterone to a human female for atleast four weeks without concurrent administration of gonadotropin. 2.The method according to claim 1, wherein between 15 mg and 40 mg of DHEAis administered three times a day to said female.
 3. The methodaccording to claim 1, wherein said androgen is DHEA, micronized,pharmaceutical grade and is orally administered.
 4. The method accordingto claim 1, wherein said method decreases miscarriage rates.
 5. Themethod according to claim 1, wherein said human female is 35 years ofage or older.
 6. The method according to claim 1, wherein said humanfemale has been diagnosed with diminished ovarian reserve.
 7. The methodaccording to claim 1, further comprising administering said androgen tothe female for at least four weeks prior to starting to an in vitrofertilization (IVF) cycle.
 8. The method according to claim 7, furthercomprising performing preimplantation genetic screening of human embryosduring in vitro fertilization to determine the number and percentage ofaneuploid embryos.
 9. The method according to claim 8, whereinpreimplantation genetic screening is performed utilizing fluorescence insitu hybridization and utilizing probes for chromosomes X, Y, 13, 16,18, 21 and
 22. 10. The method according to claim 2, whereinapproximately 25 mg of said androgen is administered three times a dayto said female.